Combination of a PD-1 antagonist and CPG-C type oligonucleotide for treating cancer

ABSTRACT

The present disclosure describes combination therapies comprising an antagonist of Programmed Death 1 receptor (PD-1) and a Toll-like receptor 9 (TLR9) agonist that is a CpG-C type oligonucleotide, and the use of the combination therapies for the treatment of cancer.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/JP2016/034275, filed May 26, 2016, and claims benefit of U.S.Provisional Application Nos. 62/168,449, filed on May 29, 2015 and62/169,309, filed Jun. 1, 2015.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 26, 2016, isnamed 24127_SL.txt and is 65,000 bytes in size.

FIELD OF THE INVENTION

The present invention relates to combination therapies useful for thetreatment of cancer. In particular, the invention relates to acombination therapy which comprises an antagonist of a Programmed Death1 protein (PD-1) and a CpG-C type oligonucleotide, which is a Toll-likereceptor 9 (TLR9) agonist.

BACKGROUND OF THE INVENTION

PD-1 is recognized as an important molecule in immune regulation and themaintenance of peripheral tolerance. PD-1 is moderately expressed onnaive T, B and NKT cells and up-regulated by TB cell receptor signalingon lymphocytes, monocytes and myeloid cells (1).

Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), areexpressed in human cancers arising in various tissues. In large samplesets of e.g. ovarian, renal, colorectal, pancreatic, liver cancers andmelanoma, it was shown that PD-L1 expression correlated with poorprognosis and reduced overall survival irrespective of subsequenttreatment (2-13). Similarly, PD-1 expression on tumor infiltratinglymphocytes was found to mark dysfunctional T cells in breast cancer andmelanoma (14-15) and to correlate with poor prognosis in renal cancer(16). Thus, it has been proposed that PD-L1 expressing tumor cellsinteract with PD-1 expressing T cells to attenuate T cell activation andevasion of immune surveillance, thereby contributing to an impairedimmune response against the tumor.

Several monoclonal antibodies that inhibit the interaction between PD-1and one or both of its ligands PD-L1 and PD-L2 are in clinicaldevelopment for treating cancer. It has been proposed that the efficacyof such antibodies might be enhanced if administered in combination withother approved or experimental cancer therapies, e.g., radiation,surgery, chemotherapeutic agents, targeted therapies, agents thatinhibit other signaling pathways that are disregulated in tumors, andother immune enhancing agents.

Administration of certain DNA sequences, generally known asimmunostimulatory sequences, induces an immune response with a Th1-typebias as indicated by secretion of Th1-associated cytokines.Administration of an immunostimulatory polynucleotide with an antigenresults in a Th1-type immune response to the administered antigen. Romanet al. (1997) Nature Med. 3:849-854. For example, mice injectedintradermally with Escherichia coli (E. coli) β-galactosidase (β-Gal) insaline or in the adjuvant alum responded by producing specific IgG1 andIgE antibodies, and CD4⁺ cells that secreted IL-4 and IL-5, but notIFN-γ, demonstrating that the T cells were predominantly of the Th2subset. However, mice injected intradermally (or with a tyne skinscratch applicator) with plasmid DNA (in saline) encoding 13-Gal andcontaining an immunostimulatory sequence responded by producing IgG2aantibodies and CD4⁺ cells that secreted IFN-γ, but not IL-4 and IL-5,demonstrating that the T cells were predominantly of the Th1 subset.Moreover, specific IgE production by the plasmid DNA-injected mice wasreduced 66-75%. Raz et al. (1996) Proc. Natl. Acad. Sci. USA93:5141-5145. In general, the response to naked DNA immunization ischaracterized by production of IL-2, TNFα and IFN-γ byantigen-stimulated CD4⁺ T cells, which is indicative of a Th1-typeresponse. This is particularly important in treatment of allergy andasthma as shown by the decreased IgE production. The ability ofimmunostimulatory polynucleotides to stimulate a Th1-type immuneresponse has been demonstrated with bacterial antigens, viral antigensand with allergens (see, for example, WO 98/55495).

There is a need in the art to improve the efficacy of cancerimmunotherapy. Therefore, it is desirable to explore combination therapyfor PD-1 antagonists and immunostimulatory oligonucleotide sequences.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for treating cancerin an individual comprising administering to the individual acombination therapy which comprises a PD-1 antagonist and a TLR9agonist, wherein the TLR9 agonist is a CpG-C type oligonucleotide.

In another embodiment, the invention provides a medicament comprising aPD-1 antagonist for use in combination with a TLR9 agonist for treatingcancer, wherein the TLR9 agonist is a CpG-C type oligonucleotide. In yetanother embodiment, the invention provides a medicament comprising aTLR9 agonist for use in combination with a PD-1 antagonist for treatingcancer, wherein the TLR9 agonist is a CpG-C type oligonucleotide.

Other embodiments provide use of a PD-1 antagonist in the manufacture ofa medicament for treating cancer in an individual when administered incombination with a TLR9 agonist and use of a TLR9 agonist in themanufacture of a medicament for treating cancer in an individual whenadministered in combination with a PD-1 antagonist. In such embodiments,the TLR9 agonist is a CpG-C type oligonucleotide.

In a still further embodiment, the invention provides use of a PD-1antagonist and a TLR9 agonist in the manufacture of medicaments fortreating cancer in an individual, wherein the TLR9 agonist is a CpG-Ctype oligonucleotide. In some embodiments, the medicaments comprise akit, and the kit also comprises a package insert comprising instructionsfor using the PD-1 antagonist in combination with the TLR9 agonist totreat cancer in an individual.

In a further embodiment, the combination therapy of the methods,medicaments or kits discussed above further comprises an anti-IL-10antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of the light chain and heavy chainCDRs for an exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:1-6).

FIG. 2 shows amino acid sequences of the light chain and heavy chainCDRs for another exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:7-12).

FIG. 3 shows amino acid sequences of the heavy chain variable region andfull length heavy chain for an exemplary anti-PD-1 monoclonal antibodyuseful in the present invention (SEQ ID NO:13 and SEQ ID NO:14).

FIG. 4 shows amino acid sequences of alternative light chain variableregions for an exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:15-17).

FIG. 5 shows amino acid sequences of alternative light chains for anexemplary anti-PD-1 monoclonal antibody useful in the present invention,with FIG. 5A showing the amino acid sequences for the K09A-L-11 andK09A-L-16 light chains (SEQ ID NOs:18 and 19, respectively) and FIG. 5Bshowing the amino acid sequence for the K09A-L-17 light chain (SEQ IDNO:20).

FIG. 6 shows amino acid sequences of the heavy and light chains forpembrolizumab (SEQ ID NOs. 21 and 22, respectively).

FIG. 7 shows amino acid sequences of the heavy and light chains fornivolumab (SEQ ID NOs. 23 and 24, respectively).

FIG. 8 shows amino acid sequences of anti-IL-10 hum12G8, with lightchain sequence of SEQ ID NO: 35 and heavy chain sequence of SEQ ID NO:34.

FIG. 9 shows amino acid sequences of anti-IL-10 TC40.11D8, with lightchain sequence of SEQ ID NO: 37 and heavy chain sequence of SEQ ID NO:36.

FIG. 10 shows tumor growth of injected tumors in mouse TC-1 bilateraltumor model. Panel A shows volume of injected tumors for individualanimals and number of complete regressions (CRs) per group. Panel Bshows median volume of injected tumors with error bar indicating 68%confidence interval. Panel C compares volumes of injected tumors betweentreatment groups by day. Panel D shows unadjusted andmultiplicity-adjusted P-values for comparison of volumes of injectedtumors between treatments. Unadjusted p value refers to two-sidedp-values based on the Peto & Peto version of the Gehan-Breslownonparametric test statistic for right-censored data. P-values wereestimated from 20,000 random reassignments of animals between the twotreatments being compared. Multiplicity adjusted p-values refers top-values adjusted to control the familywise error rate across all timepoints for a given pair of treatments. Adjustment was by applying themaxT procedure of Westfall and Young to the permutation distributions.

FIG. 11 shows tumor growth of non-injected tumors in mouse TC-1bilateral tumor model. Panel A shows volume of non-injected tumors forindividual animals and number of complete regressions (CRs) per group.Panel B shows median volume of non-injected tumors with error barindicating 68% confidence interval. Panel C compares volumes ofnon-injected tumors between treatment groups by day. Panel D showsunadjusted and multiplicity-adjusted P-values for comparison of volumesof non-injected tumors between treatments. Unadjusted p value refers totwo-sided p-values based on the Peto & Peto version of the Gehan-Breslownonparametric test statistic for right-censored data. P-values wereestimated from 20,000 random reassignments of animals between the twotreatments being compared. Multiplicity adjusted p-values refers top-values adjusted to control the familywise error rate across all timepoints for a given pair of treatments. Adjustment was by applying themaxT procedure of Westfall and Young to the permutation distributions.

FIG. 12 shows the induction of IFNα2a and IL-10 in human PBMCs (2donors) with treatment of C59-08 for 24 hours.

FIG. 13 shows induction of mRNA expression of IFNα-inducible genes(Panel A), cytokines (Panel B), and immune activation markers (Panel C)in a human renal cell carcinoma histoculture following treatment withC59-08 for 24 hours.

FIG. 14A shows the distribution of tumor nodule size in mice injectedwith CT-26 colon carcinoma cells. FIG. 14B shows the number of tumorinfiltrating leukocytes (TILs) per gram of tumor tissue. Significancewas calculated using an unpaired test using Prism GraphPad software.FIG. 14C shows the levels of gene expression of various markers of Tcell infiltration and activation, while FIG. 14D shows the levels ofgene expression of various type I interferon (IFN) responsive markers intumor tissue, versus tumor size.

FIG. 15A shows the mean tumor size, FIG. 15B shows the percent survival,and FIG. 15C shows the percent survival of various groups of treated anduntreated mice, which were engrafted with CT-26 colon carcinoma cells.

FIG. 16 shows the percent survival of mice engrafted with CT-26 coloncarcinoma cells, which received either anti-PD-1 Ab systemically andC59-08 intratumorally, in the presence or absence of CD4 or CD8 cells,or were left untreated.

FIG. 17 shows the percent survival of mice engrafted bilaterally withCT-26 colon carcinoma cells, which received either anti-PD-1 Absystemically and C59-08 intratumorally, or were left untreated.

FIG. 18A shows the tumor volume of mice engrafted with CT-26 coloncarcinoma cells, which received various treatments, relative to the meantumor volume of control oligonucleotide-treated mice. FIG. 18 B showsthe percent CD8+ T cells among CD45+ tumor-infiltrating leukocytes, andthe total number of CD8+ T cells per gram of tumor tissue.

FIG. 18C and FIG. 18D shows the levels of TNF-α and IFN-γ production by150,000 tumor infiltrating leukocytes, as measured by intracellularstaining and flow cytometry gated on CD8+ T cells, after beingstimulated for 3 hours with PMA and ionomycin in the presence of BFA(scattered dash bars) or BFA alone (dense dashed bars). For FIG. 18C,the numbers labeled on the Y axis are −10³, 0, 10³, 10⁴, 10⁵ from bottomto top, respectively, and the numbers labeled on the X axis are −10³, 0,10³, 10⁴, 10⁵ from left to right, respectively.

FIG. 19A shows the tumor growth curve of mice engrafted with CT-26 coloncarcinoma cells, which received either C59-08 intratumorally, or acontrol oligonucleotide intratumorally. FIG. 19B shows the levels ofexpression of various type I interferon responsive genes by tumorinfiltrating leukocytes of mice treated with either C59-08 or a controloligonucleotide. Data represented as relative threshold cycle (CT) ofthe gene of interest relative to the housekeeping gene, ubiquitin.

DETAILED DESCRIPTION Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

BOR Best overall response

BID One dose twice daily

CBR Clinical Benefit Rate

CDR Complementarity determining region

CHO Chinese hamster ovary

CR Complete Response

DCR Disease Control Rate

DFS Disease free survival

DLT Dose limiting toxicity

DOR Duration of Response

DSDR Durable Stable Disease Rate

FFPE Formalin-fixed, paraffin-embedded

FR Framework region

IgG Immunoglobulin G

IHC Immunohistochemistry or immunohistochemical

irRC Immune related response criteria

IV Intravenous

MTD Maximum tolerated dose

NCBI National Center for Biotechnology Information

NCI National Cancer Institute

ORR Objective response rate

OS Overall survival

PD Progressive disease

PD-1 Programmed Death 1

PD-L1 Programmed Cell Death 1 Ligand 1

PD-L2 Programmed Cell Death 1 Ligand 2

PFS Progression free survival

PR Partial response

Q2W One dose every two weeks

Q3W One dose every three weeks

QD One dose per day

RECIST Response Evaluation Criteria in Solid Tumors

SD Stable disease

VH Immunoglobulin heavy chain variable region

VK Immunoglobulin kappa light chain variable region

I. Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administration” as it applies to an animal, human, experimentalsubject, cell, tissue, organ, or biological fluid, refers to contact ofan exogenous pharmaceutical, therapeutic, diagnostic agent, orcomposition to the animal, human, subject, cell, tissue, organ, orbiological fluid. Treatment of a cell encompasses contact of a reagentto the cell, as well as contact of a reagent to a fluid, where the fluidis in contact with the cell. The term “subject” includes any organism,preferably an animal, more preferably a mammal (e.g., rat, mouse, dog,cat, rabbit) and most preferably a human.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological or binding activity. Thus, it is used inthe broadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), humanized, fully human antibodies, chimeric antibodies andcamelized single domain antibodies. “Parental antibodies” are antibodiesobtained by exposure of an immune system to an antigen prior tomodification of the antibodies for an intended use, such as humanizationof an antibody for use as a human therapeutic.

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of the heavy chain maydefine a constant region primarily responsible for effector function.Typically, human light chains are classified as kappa and lambda lightchains. Furthermore, human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids. See generally, FundamentalImmunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), which are located within relativelyconserved framework regions (FR). The CDRs are usually aligned by theframework regions, enabling binding to a specific epitope. In general,from N-terminal to C-terminal, both light and heavy chains variabledomains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignmentof amino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, unless otherwise indicated, “antibody fragment” or“antigen binding fragment” refers to antigen binding fragments ofantibodies, i.e. antibody fragments that retain the ability to bindspecifically to the antigen bound by the full-length antibody, e.g.fragments that retain one or more CDR regions. Examples of antibodybinding fragments include, but are not limited to, Fab, Fab′, F(ab)₂,and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules, e.g., sc-Fv; nanobodies and multispecific antibodies formedfrom antibody fragments.

An antibody that “specifically binds to” a specified target protein isan antibody that exhibits preferential binding to that target ascompared to other proteins, but this specificity does not requireabsolute binding specificity. An antibody is considered “specific” forits intended target if its binding is determinative of the presence ofthe target protein in a sample, e.g. without producing undesired resultssuch as false positives. Antibodies, or binding fragments thereof,useful in the present invention will bind to the target protein with anaffinity that is at least two fold greater, preferably at least tentimes greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with non-targetproteins. As used herein, an antibody is said to bind specifically to apolypeptide comprising a given amino acid sequence, e.g. the amino acidsequence of a mature human PD-1 or human PD-L1 molecule, if it binds topolypeptides comprising that sequence but does not bind to proteinslacking that sequence.

“Chimeric antibody” refers to an antibody in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in an antibody derived from a particular species(e.g., human) or belonging to a particular antibody class or subclass,while the remainder of the chain(s) is identical with or homologous tocorresponding sequences in an antibody derived from another species(e.g., mouse) or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

“Human antibody” refers to an antibody that comprises humanimmunoglobulin protein sequences only. A human antibody may containmurine carbohydrate chains if produced in a mouse, in a mouse cell, orin a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or“rat antibody” refer to an antibody that comprises only mouse or ratimmunoglobulin sequences, respectively.

“Humanized antibody” refers to forms of antibodies that containsequences from non-human (e.g., murine) antibodies as well as humanantibodies. Such antibodies contain minimal sequence derived fromnon-human immunoglobulin. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. The prefix “hum”, “hu” or “h” is added to antibodyclone designations when necessary to distinguish humanized antibodiesfrom parental rodent antibodies. The humanized forms of rodentantibodies will generally comprise the same CDR sequences of theparental rodent antibodies, although certain amino acid substitutionsmay be included to increase affinity, increase stability of thehumanized antibody, or for other reasons.

“Anti-tumor response” when referring to a cancer patient treated with atherapeutic regimen, such as a combination therapy described herein,means at least one positive therapeutic effect, such as for example,reduced number of cancer cells, reduced tumor size, reduced rate ofcancer cell infiltration into peripheral organs, reduced rate of tumormetastasis or tumor growth, or progression free survival. Positivetherapeutic effects in cancer can be measured in a number of ways (See,W. A. Weber, J. Null. Med. 50:1S-10S (2009); Eisenhauer et al., supra).In some embodiments, an anti-tumor response to a combination therapydescribed herein is assessed using RECIST 1.1 criteria, bidimentionalirRC or unidimensional irRC. In some embodiments, an anti-tumor responseis any of SD, PR, CR, PFS, or DFS.

“Bidimensional irRC” refers to the set of criteria described in WolchokJ D, et al. Guidelines for the evaluation of immune therapy activity insolid tumors: immune-related response criteria. Clin Cancer Res. 2009;15(23):7412-7420. These criteria utilize bidimensional tumormeasurements of target lesions, which are obtained by multiplying thelongest diameter and the longest perpendicular diameter (cm²) of eachlesion.

“Biotherapeutic agent” means a biological molecule, such as an antibodyor fusion protein, that blocks ligand/receptor signaling in anybiological pathway that supports tumor maintenance and/or growth orsuppresses the anti-tumor immune response. Classes of biotherapeuticagents include, but are not limited to, antibodies to VEGF, EGFR,Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4,OX-40, 4-1BB, and ICOS.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma)colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma), breast; Hematologic: blood (myeloidleukemia [acute and chronic], acute lymphoblastic leukemia, chroniclymphocytic leukemia, myeloproliferative diseases, multiple myeloma,myelodysplastic syndrome), Hodgkin's disease, non Hodgkin's lymphoma[malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi,lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:neuroblastoma. In another embodiment, the cancer is carcinoma, lymphoma,leukemia, blastoma, and sarcoma. More particular examples of suchcancers include squamous cell carcinoma, myeloma, small-cell lungcancer, non-small cell lung cancer, glioma, hodgkin's lymphoma,non-hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma,gastrointestinal (tract) cancer, renal cancer, ovarian cancer, livercancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastomamultiforme, cervical cancer, brain cancer, stomach cancer, bladdercancer, hepatoma, breast cancer, colon carcinoma, and head and neckcancer. Another particular example of cancer includes renal cellcarcinoma. Yet another particular example of cancer is non-hodgkin'slymphoma or cutaneous T-cell lymphoma. Yet another particular example ofcancer is acute myeloid leukemia (AML) or myelodysplastic syndrome.Cancers that may be treated in accordance with the present inventioninclude those characterized by elevated expression of one or both ofPD-L1 and PD-L2 in tested tissue samples.

“CpG-C ONs” or “CpG-C type oligonucleotides” are oligonucleotides from12 to 100 bases in length, which have one or more 5′-TCG trinucleotideswherein the 5′-T is positioned 0, 1, 2, or 3 bases from the 5′-end ofthe oligonucleotide, and at least one palindromic sequence of at least 8bases in length comprising one or more unmethylated CG dinucleotides.The one or more 5′-TCG trinucleotide sequence may be separated from the5′-end of the palindromic sequence by 0, 1, or 2 bases or thepalindromic sequence may contain all or part of the one or more 5′-TCGtrinucleotide sequence. In one embodiment, the oligonucleotide is anoligodeoxynucleotide (ODN). In one embodiment, the oligonucleotide is a2′-oligodeoxynucleotide. CpG-C ODNs have the ability to stimulate Bcells, induce plasmacytoid dendritic cell (PDC) maturation and causesecretion of high levels of type I interferons (e.g., IFN-α, IFN-γ,etc.). In some embodiments, the CpG-C ODNs are 12 to 100 bases inlength, preferably 12 to 50 bases in length, preferably 12 to 40 basesin length, or preferably 12-30 bases in length. In some embodiments, theODN is at least (lower limit) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80,or 90 bases in length. In some embodiments, the ODN is at most (upperlimit) 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 bases in length. In someembodiments, the at least one palindromic sequence is 8 to 97 bases inlength, preferably 8 to 50 bases in length, or preferably 8 to 32 basesin length. In some embodiments, the at least one palindromic sequence isat least (lower limit) 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30bases in length. In some embodiments, the at least one palindromicsequence is at most (upper limit) 50, 48, 46, 44, 42, 40, 38, 36, 34,32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12 or 10 bases in length. In oneembodiment, the oligonucleotide is an oligodeoxynucleotide. In oneembodiment, one or more of the internucleotide linkages of the CpG-C ODNare modified linkages. In one embodiment, one or more of theinternucleotide linkages of CpG-C ODN are phosphorothioate (PS)linkages. In one embodiment, all of the internucleotide linkages ofCpG-C ODN are phosphorothioate (PS) linkages. A phosphorothioatebackbone refers to all of the internucleotide linkages of CpG-C ODNbeing phosphorothioate (PS) linkages.

The CpG-C type ODNs and SEQ ID NO: 38-51 discussed herein are in theirpharmaceutically acceptable salt form unless otherwise indicated.Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, zinc salts, salts with organic bases (forexample, organic amines) such as N-Me-D-glucamine,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline,tromethamine, dicyclohexylamines, t-butyl amines, and salts with aminoacids such as arginine, lysine and the like. In one embodiment, theCpG-C type ODNs are in the ammonium, sodium, lithium, or potassium saltform. In one preferred embodiment, the CpG-C type ODNs are in the sodiumsalt form. The CpG-C ODN may be provided in a pharmaceutical solutioncomprising a pharmaceutically acceptable excipient. Alternatively, theCpG-C ODN may provided as a lyophilized solid, which is subsequentlyreconstituted in sterile water, saline or a pharmaceutically acceptablebuffer before administration.

Pharmaceutically acceptable excipients of the present disclosure includefor instance, solvents, bulking agents, buffering agents, tonicityadjusting agents, and preservatives (see, e.g., Pramanick et al., PharmaTimes, 45:65-77, 2013). In some embodiments the pharmaceuticalcompositions may comprise an excipient that functions as one or more ofa solvent, a bulking agent, a buffering agent, and a tonicity adjustingagent (e.g., sodium chloride in saline may serve as both an aqueousvehicle and a tonicity adjusting agent). The pharmaceutical compositionsof the present disclosure are suitable for parenteral administration.

In some embodiments, the pharmaceutical compositions comprise an aqueousvehicle as a solvent. Suitable vehicles include for instance sterilewater, saline solution, phosphate buffered saline, and Ringer'ssolution. In some embodiments, the composition is isotonic.

The pharmaceutical compositions may comprise a bulking agent. Bulkingagents are particularly useful when the pharmaceutical composition is tobe lyophilized before administration. In some embodiments, the bulkingagent is a protectant that aids in the stabilization and prevention ofdegradation of the active agents during freeze or spray drying and/orduring storage. Suitable bulking agents are sugars (mono-, di- andpolysaccharides) such as sucrose, lactose, trehalose, mannitol,sorbital, glucose and raffinose.

The pharmaceutical compositions may comprise a buffering agent.Buffering agents control pH to inhibit degradation of the active agentduring processing, storage and optionally reconstitution. Suitablebuffers include for instance salts comprising acetate, citrate,phosphate or sulfate. Other suitable buffers include for instance aminoacids such as arginine, glycine, histidine, and lysine. The bufferingagent may further comprise hydrochloric acid or sodium hydroxide. Insome embodiments, the buffering agent maintains the pH of thecomposition within a range of 4 to 9. In some embodiments, the pH isgreater than (lower limit) 4, 5, 6, 7 or 8. In some embodiments, the pHis less than (upper limit) 9, 8, 7, 6 or 5. That is, the pH is in therange of from about 4 to 9 in which the lower limit is less than theupper limit.

The pharmaceutical compositions may comprise a tonicity adjusting agent.Suitable tonicity adjusting agents include for instance dextrose,glycerol, sodium chloride, glycerin and mannitol.

The pharmaceutical compositions may comprise a preservative. Suitablepreservatives include for instance antioxidants and antimicrobialagents. However, in preferred embodiments, the pharmaceuticalcomposition is prepared under sterile conditions and is in a single usecontainer, and thus does not necessitate inclusion of a preservative.

The term “palindromic sequence” or “palindrome” refers to a nucleic acidsequence that is an inverted repeat, e.g., ABCDD′C′B′A′, where thebases, e.g., A, and A′, B and B′, C and C′, D and D′, are capable offorming Watson-Crick base pairs. Such sequences may be single-strandedor may form double-stranded structures or may form hairpin loopstructures under some conditions. For example, as used herein, “an 8base palindrome” refers to a nucleic acid sequence in which thepalindromic sequence is 8 bases in length, such as ABCDD′C′B′A′. Apalindromic sequence may be part of an oligonucleotide that alsocontains non-palindromic sequences. An oligonucleotide may contain oneor more palindromic sequence portions and one or more non-palindromicsequence portions. Alternatively, an oligonucleotide sequence may beentirely palindromic. In an oligonucleotide with more than onepalindromic sequence portion, the palindromic sequence portions may ormay not overlap with each other.

In one embodiment, the CpG-C ODNs of the present disclosure comprise:

(a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z),N_(v) (SEQID NO:38) wherein N are nucleosides, x=0, 1, 2 or 3, y=1, 2, 3 or 4,w=0, 1 or 2, p=0 or 1, q=0, 1 or 2, v=0 to 89 and z=1 to 20, X₁ and X₁′are self-complementary nucleosides, X₂ and X₂′ are self-complementarynucleosides, and wherein the 5′-T of the (TCG(N_(q)))_(y), sequence is0-3 bases from the 5′ end of the oligonucleotide; and(b) a palindromic sequence at least 8 bases in length wherein thepalindromic sequence comprises the first (X₁X₂CGX₂′X₁′) (SEQ ID NO:55)of the (X₁X₂CGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO: 56) sequences, wherein theODN is from 12 to 100 bases in length. In some embodiments, x=0, y=1,w=0, p=0 or 1, q=0, 1 or 2, v=0 to 20 and z=1, 2, 3 or 4. In someembodiments, X₁ and X₂ are each either A or T. In some embodiments, thepalindromic sequence has a base composition of more than one-third Asand Ts. In some embodiments, the CpG-C ODN comprises a sequence selectedfrom the group consisting of SEQ ID NOs:38-51.

In some embodiments, the CpG-C ODNs of the present disclosure consist ofTCGN_(q)(X₁X₂CGX₂′X₁′CG)_(z)N_(v) (SEQ ID NO:39), wherein N arenucleosides, q=0, 1, 2, 3, 4, or 5, v=0 to 20, z=1 to 4, X₁ and X₁′ areself-complementary nucleosides, X₂ and X₂′ are self-complementarynucleosides, and wherein the ODN is at least 12 bases in length. In someembodiments, the CpG-C ODN consists of a sequence selected from thegroup consisting of SEQ ID NOs:38-51.

In some embodiments, the CpG-C ODNs of the present disclosure consist of5′-TCGN_(q)TTCGAACGTTCGAACGTTN_(s)-3′ (SEQ ID NO:40), wherein N arenucleosides, q=0, 1, 2, 3, 4, or 5, s=0 to 20, and wherein the ODN is atleast 12 bases in length. In one embodiment, s=0, 1, 2, 3, 4, or 5. Insome embodiments, the CpG-C ODN consists of a sequence selected from thegroup consisting of

(SEQ ID NO: 42)  5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ q  = 0 and s = 4,(SEQ ID NO: 43) 5′-TCGAACGTTCGAACGTTCGAACGTT-3′ q = 4 and s = 0,(SEQ ID NO: 45) 5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ q = 4 and s = 5,(SEQ ID NO: 46) 5′-TCGTAACGTTCGAACGTTCGAACGTTA-3′ q = 5 and s = 1, and(SEQ ID NO: 47) 5′-TCGTAACGTTCGAACGTTCGAACGTT-3′ q  = 5 and s = 0.

In one embodiment, the TLR9 agonist is a CpG-C ODN consisting of thesequence 5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO:45). In anotherembodiment, the CpG-C ODN is the sodium salt of5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO:45). In a furtherembodiment, the CpG-C type oligonucleotide has a sequence that consistsof 5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ (SEQ ID NO:42). In a furtherembodiment, the CpG-C type oligonucleotide is a sodium salt of5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ (SEQ ID NO:42).

In another embodiment, the TLR9 agonist CpG-C type oligonucleotide isselected from the group consisting of:

(SEQ ID NO: 41) 5′-TCGTCGAACGTTCGAGATGAT-3′; (SEQ ID NO: 42)5′-TCGTTCGAACGTTCGAACGTTCGAA-3′; (SEQ ID NO: 43)5′-TCGAACGTTCGAACGTTCGAACGTT-3′; (SEQ ID NO: 44)5′-TCGAACGTTCGAACGTTCGAATTTT-3′; (SEQ ID NO: 45)5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′; (SEQ ID NO: 46)5′-TCGTAACGTTCGAACGTTCGAACGTTA-3′; (SEQ ID NO: 47)5′-TCGTAACGTTCGAACGTTCGAACGTT-3′; (SEQ ID NO: 48)5′-TCGTAACGTTCGAACGTTCGAACGT-3′; (SEQ ID NO: 49)5′-TCGTAACGTTCGAACGTTCGAACG-3′; (SEQ ID NO: 50)5′-TCGTAACGTTCGAACGTTCGAAC-3′; and (SEQ ID NO: 51)5′-TCGTAACGTTCGAACGTTCGAA-3′.

TABLE 1 Motif and Sequences of CpG-C type oligonucleotides SEQ CompoundID # NO: Sequence C59-01 385′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z) N_(v)-3′ C59-0239 5′-TCGN_(q)(X₁X₂CGX₂′X₁′CG)_(z)N_(v)-3′ C59-03 405′-TCGN_(q)TTCGAACGTTCGAACGTTN_(s)-3′ C59-04 415′-TCGTCGAACGTTCGAGATGAT-3′ C59-05 42 5′-TCGTTCGAACGTTCGAACGTTCGAA-3′C59-06 43 5′-TCGAACGTTCGAACGTTCGAACGTT-3′ C59-07 445′-TCGAACGTTCGAACGTTCGAATTTT-3′ C59-08 455′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ C59-09 465′-TCGTAACGTTCGAACGTTCGAACGTTA-3′ C59-10 475′-TCGTAACGTTCGAACGTTCGAACGTT-3′ C59-11 485′-TCGTAACGTTCGAACGTTCGAACGT-3′ C59-12 49 5′-TCGTAACGTTCGAACGTTCGAACG-3′C59-13 50 5′-TCGTAACGTTCGAACGTTCGAAC-3′ C59-14 515′-TCGTAACGTTCGAACGTTCGAA-3′

It is understood that, with respect to formulae or sequence motifsdescribed herein, any and all parameters are independently selected. Forexample, if x=0-2, y may be independently selected regardless of thevalue of x (or any other selectable parameter in a formula), as long asthe total oligonucleotide length limitation is met.

Additional CpG-C oligonucleotides having sequences encompassed by themotifs of the present disclosure are suitable for use in the methods andmedicaments disclosed herein. A plurality of additional CpG-Coligonucleotides having sequences encompassed by the motifs of thepresent disclosure are described in U.S. Pat. Nos. 7,745,606, 8,158,768,and 8,871,732 to Dynavax Technologies Corporation. These sequences arehereby incorporated by reference.

CpG oligonucleotides have been described in the art and their activitymay be readily determined using standard assays, which measure variousaspects of immune responses (e.g., cytokine secretion, antibodyproduction, NK cell activation, B cell proliferation, T cellproliferation, etc.). Exemplary methods are described in WO 97/28259; WO98/16247; WO 99/11275, WO 98/55495 and WO 00/61151, as well as U.S. Pat.Nos. 7,745,606, 8,158,768, and 8,871,732 to Dynavax TechnologiesCorporation. Accordingly, these and other methods can be used to detectand quantify immunomodulatory activity of CpG oligonucleotides.

CpG-C oligonucleotides may contain modifications. Suitable modificationsinclude but are not limited to, modifications of the 3′OH or 5′OH group,modifications of the nucleotide base, modifications of the sugarcomponent, and modifications of the phosphate group. Modified bases maybe included in the palindromic sequence as long as the modified base(s)maintains the same specificity for its natural complement throughWatson-Crick base pairing (e.g., the palindromic portion of the CpG-Coligonucleotide remains self-complementary).

CpG-C oligonucleotides may be linear, may be circular or includecircular portions and/or a hairpin loop. CpG-C oligonucleotides may besingle stranded or double stranded. CpG-C oligonucleotides may be DNA,RNA or a DNA/RNA hybrid.

CpG-C oligonucleotides may contain naturally-occurring or modified,non-naturally occurring bases, and may contain modified sugar,phosphate, and/or termini. For example, in addition to phosphodiesterlinkages, phosphate modifications include, but are not limited to,methyl phosphonate, phosphorothioate, phosphoramidate (bridging ornon-bridging), phosphotriester and phosphorodithioate and may be used inany combination. In some embodiments, CpG-C oligonucleotides have onlyphosphorothioate linkages, only phosphodiester linkages, or acombination of phosphodiester and phosphorothioate linkages.

Sugar modifications known in the field, such as 2′-alkoxy-RNA analogs,2′-amino-RNA analogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNAchimeras and others described herein, may also be made and combined withany phosphate modification. Examples of base modifications include butare not limited to addition of an electron-withdrawing moiety to C-5and/or C-6 of a cytosine of the CpG-C oligonucleotide (e.g.,5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) andC-5 and/or C-6 of a uracil of the CpG-C oligonucleotide (e.g.,5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). As notedabove, use of a base modification in a palidromic sequence of a CpG-Coligonucleotide should not interfere with the self-complementarity ofthe bases involved for Watson-Crick base pairing. However, outside of apalindromic sequence, modified bases may be used without thisrestriction. For instance, 2′-O-methyl-uridine and 2′-O-methyl-cytidinemay be used outside of the palindromic sequence, whereas,5-bromo-2′-deoxycytidine may be used both inside and outside thepalindromic sequence. Other modified nucleotides, which may be employedboth inside and outside of the palindromic sequence include7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

Duplex (i.e., double stranded) and hairpin forms of mostoligonucleotides are in dynamic equilibrium, with the hairpin formgenerally favored at low oligonucleotide concentration and highertemperatures. Covalent interstrand or intrastrand cross-links increaseduplex or hairpin stability, respectively, towards thermal-, ionic-,pH-, and concentration-induced conformational changes. Chemicalcross-links can be used to lock the polynucleotide into either theduplex or the hairpin form for physicochemical and biologicalcharacterization. Cross-linked oligonucleotides that areconformationally homogeneous and are “locked” in their most active form(either duplex or hairpin form) could potentially be more active thantheir uncross-linked counterparts. Accordingly, some CpG-Coligonucleotides of the present disclosure contain covalent interstrandand/or intrastrand cross-links.

A variety of ways to chemically cross-link duplex DNA are known in theart. Any cross-linking method may be used as long as the cross-linkedpolynucleotide product possesses the desired immunomodulatory activity.One method, for example, results in a disulfide bridge between twoopposing thymidines at the terminus of the duplex or hairpin. For thiscross-linking method, the oligonucleotide(s) of interest is synthesizedwith a 5′-DMT-N3-(tBu-SS-ethyl)thymidine-3′-phosphoramidite (“T*”). Toform the disulfide bridge, the mixed disulfide bonds are reduced,oligonucleotide purified, the strands hybridized and the compoundair-oxidized to form the intrastrand cross-link in the case of a hairpinform or the interstrand cross-link in the case of a duplex form.Alternatively, the oligonucleotides may be hybridized first and thenreduced, purified and air-oxidized. Such methods and others aredescribed in the art (see, e.g., Glick et al., J Org Chem, 56:6746-6747,1991, Glick et al., J Am Chem Soc, 114:5447-5448, 1992, Goodwin et al.,Tetrahedron Letters 35:1647-1650, 1994, Wang et al., J Am Chem Soc,117:2981-2991, 1995, Osborne et al., Bioorganic & Medicinal ChemistryLetters, 6:2339-2342, 1996 and Osborne et al., J Am Chem Soc,118:11993-12003, 1996).

Another cross-linking method forms a disulfide bridge between offsetresidues in the duplex or hairpin structure. For this cross-linkingmethod, the oligonucleotide(s) of interest is synthesized withconvertible nucleosides (commercially available, for example, from GlenResearch). This method utilizes, for example, an A-A disulfide or a C-Adisulfide bridge and linkages through other bases are also possible. Toform the disulfide-modified polynucleotide, the polynucleotidecontaining the convertible nucleoside is reacted with cystamine (orother disulfide-containing amine). To form the disulfide bridge, themixed disulfide bonds are reduced, oligonucleotide purified, the strandshybridized and the compound air-oxidized to form the intrastrandcross-link in the case of a hairpin form or the interstrand cross-linkin the case of a duplex form. Alternatively, the oligonucleotides may behybridized first and then reduced, purified and air-oxidized. Suchmethods are described in the art (see, e.g., Ferentz et al., J Am ChemSoc, 113:4000-4002, 1991, and Ferentz et al., J Am Chem Soc,115:9006-9014, 1993).

The techniques for making polynucleotides and modified polynucleotidesare known in the art. Naturally occurring DNA or RNA, containingphosphodiester linkages, is generally synthesized by sequentiallycoupling the appropriate nucleoside phosphoramidite to the 5′-hydroxygroup of the growing oligonucleotide attached to a solid support at the3′-end, followed by oxidation of the intermediate phosphite triester toa phosphate triester. Once the desired polynucleotide sequence has beensynthesized, the polynucleotide is removed from the support, thephosphate triester groups are deprotected to phosphate diesters and thenucleoside bases are deprotected using aqueous ammonia or other bases(see, e.g., Beaucage “Oligodeoxyribonucleotide Synthesis” in Protocolsfor Oligonucleotides and Analogs, Synthesis and Properties (Agrawal,ed.) Humana Press, Totowa, N.J., 1993; Warner et al., DNA 3:401, 1984and U.S. Pat. No. 4,458,066).

The CpG-C oligonucleotide may contain phosphate-modifiedoligonucleotides, some of which are known to stabilize theoligonucleotide. Accordingly, some embodiments include stabilized CpG-Coligonucleotides. Synthesis of oligonucleotides containing modifiedphosphate linkages or non-phosphate linkages is also known in the art(see, e.g., Matteucci “Oligonucleotide Analogs: an Overview” inOligonucleotides as Therapeutic Agents, (D. J. Chadwick and G. Cardew,ed.) John Wiley and Sons, New York, N.Y., 1997). The phosphorousderivative (or modified phosphate group), which can be attached to thesugar or sugar analog moiety in the oligonucleotide, can be amonophosphate, diphosphate, triphosphate, alkylphosphonate,phosphorothioate, phosphorodithioate, phosphoramidate or the like. Thepreparation of the above-noted phosphate analogs, and theirincorporation into nucleotides, modified nucleotides andoligonucleotides, per se, has already been well described (see, e.g.,Peyrottes et al., Nucleic Acids Res, 24:1841-1848, 1996; Chaturvedi etal., Nucleic Acids Res, 24:2318-2323, 1996; and Schultz et al., NucleicAcids Res, 24:2966-2973, 1996). For example, synthesis ofphosphorothioate oligonucleotides is similar to that described above fornaturally occurring oligonucleotides except that the oxidation step isreplaced by a sulfurization step (Zon “OligonucleosidePhosphorothioates” in Protocols for Oligonucleotides and Analogs,Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190,1993).

CpG-C oligonucleotides can comprise one or more ribonucleotides(containing ribose as the only or principal sugar component),deoxyribonucleotides (containing deoxyribose as the principal sugarcomponent), modified sugars or sugar analogs. Thus, in addition toribose and deoxyribose, the sugar moiety can be pentose, deoxypentose,hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugaranalog cyclopentyl group. The sugar can be in pyranosyl or in afuranosyl form. In the CpG-C oligonucleotide, the sugar moiety ispreferably the furanoside of ribose, deoxyribose, arabinose or2′-0-alkylribose, and the sugar can be attached to the respectiveheterocyclic bases either in anomeric configuration. Sugar modificationsinclude, but are not limited to, 2′-alkoxy-RNA analogs, 2′-amino-RNAanalogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNA chimeras. Forexample, a sugar modification in the CpG-C oligonucleotide includes, butis not limited to, 2′-O-methyl-uridine and 2′-O-methyl-cytidine. Thepreparation of these sugars or sugar analogs and the respectivenucleosides wherein such sugars or analogs are attached to aheterocyclic base (nucleic acid base) per se is known, and thereforeneed not be described here. Sugar modifications may also be made andcombined with any phosphate modification in the preparation of a CpG-Coligonucleotide.

The heterocyclic bases, or nucleic acid bases, which are incorporated inthe CpG-C oligonucleotide can be the naturally-occurring principalpurine and pyrimidine bases, (namely uracil, thymine, cytosine, adenineand guanine, as mentioned above), as well as naturally-occurring andsynthetic modifications of said principal bases. Thus, a CpG-Coligonucleotide may include one or more of inosine, 2′-deoxyuridine, and2-amino-2′-deoxyadenosine.

“CBR” or “Clinical Benefit Rate” means CR+PR+durable SD.

“CDR” or “CDRs” as used herein means complementarity determiningregion(s) in a immunoglobulin variable region, defined using the Kabatnumbering system, unless otherwise indicated.

“Chemotherapeutic agent” is a chemical compound useful in the treatmentof cancer. Classes of chemotherapeutic agents include, but are notlimited to: alkylating agents, antimetabolites, kinase inhibitors,spindle poison plant alkaloids, cytoxic/antitumor antibiotics,topisomerase inhibitors, photosensitizers, anti-estrogens and selectiveestrogen receptor modulators (SERMs), anti-progesterones, estrogenreceptor down-regulators (ERDs), estrogen receptor antagonists,leutinizing hormone-releasing hormone agonists, anti-androgens,aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and anti-senseoligonucleotides that inhibit expression of genes implicated in abnormalcell proliferation or tumor growth. Chemotherapeutic agents useful inthe treatment methods of the present invention include cytostatic and/orcytotoxic agents.

“Chothia” as used herein means an antibody numbering system described inAl-Lazikani et al., JMB 273:927-948 (1997).

“Comprising” or variations such as “comprise”, “comprises” or “comprisedof” are used throughout the specification and claims in an inclusivesense, i.e., to specify the presence of the stated features but not topreclude the presence or addition of further features that maymaterially enhance the operation or utility of any of the embodiments ofthe invention, unless the context requires otherwise due to expresslanguage or necessary implication.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity or other desired property of the protein, such asantigen affinity and/or specificity. Those of skill in this artrecognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. (1987) Molecular Biologyof the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). Inaddition, substitutions of structurally or functionally similar aminoacids are less likely to disrupt biological activity. Exemplaryconservative substitutions are set forth in Table 2 below.

TABLE 2 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

“Consists essentially of,” and variations such as “consist essentiallyof” or “consisting essentially of,” as used throughout the specificationand claims, indicate the inclusion of any recited elements or group ofelements, and the optional inclusion of other elements, of similar ordifferent nature than the recited elements, that do not materiallychange the basic or novel properties of the specified dosage regimen,method, or composition. As a non-limiting example, a PD-1 antagonistthat consists essentially of a recited amino acid sequence may alsoinclude one or more amino acids, including substitutions of one or moreamino acid residues, which do not materially affect the properties ofthe binding compound.

“DCR” or “Disease Control Rate” means CR+PR+SD.

“Diagnostic anti-PD-L monoclonal antibody” means a mAb whichspecifically binds to the mature form of the designated PD-L (PD-L1 orPDL2) that is expressed on the surface of certain mammalian cells. Amature PD-L lacks the presecretory leader sequence, also referred to asleader peptide The terms “PD-L” and “mature PD-L” are usedinterchangeably herein, and shall be understood to mean the samemolecule unless otherwise indicated or readily apparent from thecontext.

As used herein, a diagnostic anti-human PD-L1 mAb or an anti-hPD-L1 mAbrefers to a monoclonal antibody that specifically binds to mature humanPD-L1. A mature human PD-L1 molecule consists of amino acids 19-290 ofthe following sequence:

(SEQ ID NO: 25) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET.

Specific examples of diagnostic anti-human PD-L1 mAbs useful asdiagnostic mAbs for immunohistochemistry (IHC) detection of PD-L1expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissuesections are antibody 20C3 and antibody 22C3, which are described in thecopending international patent application PCT/US13/075932, filed 18Dec. 2013 and published as WO2014/100079 on 26 Jun. 2014. Anotheranti-human PD-L1 mAb that has been reported to be useful for IHCdetection of PD-L1 expression in FFPE tissue sections (Chen, B. J. etal., Clin Cancer Res 19: 3462-3473 (2013)) is a rabbit anti-human PD-L1mAb publicly available from Sino Biological, Inc. (Beijing, P.R. China;Catalog number 10084-R015).

“Anti-IL-10 antibody” means an antagonist antibody that binds IL-10 toinhibit the activity of IL-10. Alternative names or synonyms for IL-10include: Interleukin-10, cytokine synthesis inhibitor factor or CSIF.Human IL-10 amino acid sequences can be found in U.S. Pat. No.6,217,857. The amino acid sequence of the mature human IL-10 protein is

(SEQ ID NO: 52) SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFI NYIEAYMTMKIRN

Anti-IL-10 antibodies useful in any of the treatment method, medicamentsand uses of the present invention include a monoclonal antibody (mAb),or antigen binding fragment thereof, which specifically binds to IL-10,and preferably specifically binds to human IL-10. The mAb may be a humanantibody, a humanized antibody or a chimeric antibody, and may include ahuman constant region. In some embodiments, the human constant region isselected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constantregions, and in preferred embodiments, the human constant region is anIgG1 or IgG4 constant region. In some embodiments, the antigen bindingfragment is selected from the group consisting of Fab, Fab′-SH, F(ab′)₂,scFv and Fv fragments.

In some preferred embodiments of the treatment method, medicaments anduses of the present invention, the anti-IL-10 antibody is a monoclonalantibody, or antigen binding fragment thereof, which comprises: (a)light chain CDRs of SEQ ID NOs: 26, 27 and 28 and heavy chain CDRs SEQID NOs: 29, 30 and 31 of anti-IL-10 hum12G8.

In other preferred embodiments of the treatment method, medicaments anduses of the present invention, the anti-IL-10 antibody is a monoclonalantibody, or antigen binding fragment thereof, which specifically bindsto human IL-10 and comprises (a) a heavy chain variable regioncomprising SEQ ID NO:32 or a variant thereof, and (b) a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:33 or a variant thereof. A variant of aheavy chain variable region sequence is identical to the referencesequence except having up to 17 conservative amino acid substitutions inthe framework region (i.e., outside of the CDRs), and preferably hasless than ten, nine, eight, seven, six or five conservative amino acidsubstitutions in the framework region. A variant of a light chainvariable region sequence is identical to the reference sequence excepthaving up to five conservative amino acid substitutions in the frameworkregion (i.e., outside of the CDRs), and preferably has less than four,three or two conservative amino acid substitution in the frameworkregion.

Table 3 below provides a list of the amino acid sequences of exemplaryanti-IL-10 mAbs for use in the treatment method, medicaments and uses ofthe present invention, and the sequences are shown in FIGS. 8-9.

TABLE 3 EXEMPLARY ANTI-HUMAN IL-10 MONOCLONAL ANTIBODIESA. Comprises light and heavy chain CDRs of hum12G8 in U.S. Pat. No. 7662379CDRL1 SEQ ID NO: 26 KTSQNIFENLA CDRL2 SEQ ID NO: 27 YNASPLQA CDRL3SEQ ID NO: 28 HQYYSGYT CDRH1 SEQ ID NO: 29 GFTFSDYHMA CDRH2SEQ ID NO: 30 SITLDATYTYYRDSVRG CDRH3 SEQ ID NO: 31 HRGFSVWLDYB. Comprises the heavy chain variable region and light chain variable regions ofhum12G8 in U.S. Pat. No. 7662379 Heavy chain VR SEQ ID NO: 32QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYHMAWVRQAPGKGLEWVASITLDATYTYYRDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHR GFSVWLDYWGQGTLVTVSSALight chain VR SEQ ID NO: 33DIQMTQSPSSLSASVGDRVTITCKTSQNIFENLAWYQQKPGKAPKLLIYNASPLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYSGYTFGPG TKLELKRTVAAC. Comprises the heavy chain and light chain of hum12G8 in U.S. Pat. No. 7662379Heavy chain SEQ ID NO: 34 Light chain SEQ ID NO: 35D. Comprises the heavy chain and light chain of TC40.11D8 in U.S. Pat. No. 8226947Heavy chain SEQ ID NO: 36 Light chain SEQ ID NO: 37

As used herein, an “anti-IL-10 hum 12G8 variant” means a monoclonalantibody which comprises heavy chain and light chain sequences that areidentical to those in anti-IL-10 hum 12G8, except for having three, twoor one conservative amino acid substitutions at positions that arelocated outside of the light chain CDRs and six, five, four, three, twoor one conservative amino acid substitutions that are located outside ofthe heavy chain CDRs, e.g., the variant positions are located in the FRregions or the constant region. In other words, anti-IL-10 hum 12G8 andan anti-IL-10 hum 12G8 variant comprise identical CDR sequences, butdiffer from each other due to having a conservative amino acidsubstitution at no more than three or six other positions in their fulllength light and heavy chain sequences, respectively. An anti-IL-10 hum12G8 variant is substantially the same as anti-IL-10 hum 12G8 withrespect to the following properties: binding affinity to IL-10 andneutralizing effect in vivo.

“DSDR” or “Durable Stable Disease Rate” means SD for ≥23 weeks.

“Framework region” or “FR” as used herein means the immunoglobulinvariable regions excluding the CDR regions.

“Kabat” as used herein means an immunoglobulin alignment and numberingsystem pioneered by Elvin A. Kabat ((1991) Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.).

“Monoclonal antibody” or “mAb” or “Mab”, as used herein, refers to apopulation of substantially homogeneous antibodies, i.e., the antibodymolecules comprising the population are identical in amino acid sequenceexcept for possible naturally occurring mutations that may be present inminor amounts. In contrast, conventional (polyclonal) antibodypreparations typically include a multitude of different antibodieshaving different amino acid sequences in their variable domains,particularly their CDRs, which are often specific for differentepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256: 495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J.Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. AllergyClin. Immunol. 116:731.

“Non-responder patient”, when referring to a specific anti-tumorresponse to treatment with a combination therapy described herein, meansthe patient did not exhibit the anti-tumor response.

“ORR” or “objective response rate” refers in some embodiments to CR+PR,and ORR_((week 24)) refers to CR and PR measured using irRECIST in eachpatient in a cohort after 24 weeks of treatment with CpG-C typeoligonucleotide in combination with pembrolizumab.

“Patient” or “subject” refers to any single subject for which therapy isdesired or that is participating in a clinical trial, epidemiologicalstudy or used as a control, including humans and mammalian veterinarypatients such as cattle, horses, dogs, and cats.

“PD-1 antagonist” means any chemical compound or biological moleculethat blocks binding of PD-L1 expressed on a cancer cell to PD-1expressed on an immune cell (T cell, B cell or NKT cell) and preferablyalso blocks binding of PD-L2 expressed on a cancer cell to theimmune-cell expressed PD-1. Alternative names or synonyms for PD-1 andits ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1,PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC,Btdc and CD273 for PD-L2. In any of the treatment method, medicamentsand uses of the present invention in which a human individual is beingtreated, the PD-1 antagonist blocks binding of human PD-L1 to humanPD-1, and preferably blocks binding of both human PD-L1 and PD-L2 tohuman PD-1. Human PD-1 amino acid sequences can be found in NCBI LocusNo.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be foundin NCBI Locus No.: NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in the any of the treatment method, medicamentsand uses of the present invention include a monoclonal antibody (mAb),or antigen binding fragment thereof, which specifically binds to PD-1 orPD-L1, and preferably specifically binds to human PD-1 or human PD-L1.The mAb may be a human antibody, a humanized antibody or a chimericantibody, and may include a human constant region. In some embodimentsthe human constant region is selected from the group consisting of IgG1,IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, thehuman constant region is an IgG1 or IgG4 constant region. In someembodiments, the antigen binding fragment is selected from the groupconsisting of Fab, Fab′-SH, F(ab′)₂, scFv and Fv fragments.

Examples of mAbs that bind to human PD-1, and useful in the treatmentmethod, medicaments and uses of the present invention, are described inU.S. Pat. Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, 8,168,757,WO2004/004771, WO2004/072286, WO2004/056875, and US2011/0271358.Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in thetreatment method, medicaments and uses of the present invention include:pembrolizumab (also known as MK-3475), a humanized IgG4 mAb with thestructure described in WHO Drug Information, Vol. 27, No. 2, pages161-162 (2013) and which comprises the heavy and light chain amino acidsequences shown in FIG. 6; nivolumab (BMS-936558), a human IgG4 mAb withthe structure described in WHO Drug Information, Vol. 27, No. 1, pages68-69 (2013) and which comprises the heavy and light chain amino acidsequences shown in FIG. 7; the humanized antibodies h409A11, h409A16 andh409A17, which are described in WO2008/156712, and AMP-514, which isbeing developed by MedImmune.

Examples of mAbs that bind to human PD-L1, and useful in the treatmentmethod, medicaments and uses of the present invention, are described inWO2013/019906, WO2010/077634 A1 and U.S. Pat. No. 8,383,796. Specificanti-human PD-L1 mAbs useful as the PD-1 antagonist in the treatmentmethod, medicaments and uses of the present invention include MPDL3280A,BMS-936559, MEDI4736, MSB0010718C and an antibody which comprises theheavy chain and light chain variable regions of SEQ ID NO:24 and SEQ IDNO:21, respectively, of WO2013/019906.

Other PD-1 antagonists useful in the treatment method, medicaments anduses of the present invention include an immunoadhesin that specificallybinds to PD-1 or PD-L1, and preferably specifically binds to human PD-1or human PD-L1, e.g., a fusion protein containing the extracellular orPD-1 binding portion of PD-L1 or PD-L2 fused to a constant region suchas an Fc region of an immunoglobulin molecule. Examples ofimmunoadhesion molecules that specifically bind to PD-1 are described inWO2010/027827 and WO2011/066342. Specific fusion proteins useful as thePD-1 antagonist in the treatment method, medicaments and uses of thepresent invention include AMP-224 (also known as B7-DCIg), which is aPD-L2-FC fusion protein and binds to human PD-1.

In some preferred embodiments of the treatment method, medicaments anduses of the present invention, the PD-1 antagonist is a monoclonalantibody, or antigen binding fragment thereof, which comprises: (a)light chain CDRs SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs SEQ ID NOs:4, 5 and 6; or (b) light chain CDRs SEQ ID NOs: 7, 8 and 9 and heavychain CDRs SEQ ID NOs: 10, 11 and 12.

In other preferred embodiments of the treatment method, medicaments anduses of the present invention, the PD-1 antagonist is a monoclonalantibody, or antigen binding fragment thereof, which specifically bindsto human PD-1 and comprises (a) a heavy chain variable region comprisingSEQ ID NO:13 or a variant thereof, and (b) a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:15 or a variant thereof; SEQ ID NO:16 or a variant thereof;and SEQ ID NO: 17 or a variant thereof. A variant of a heavy chainvariable region sequence is identical to the reference sequence excepthaving up to 17 conservative amino acid substitutions in the frameworkregion (i.e., outside of the CDRs), and preferably has less than ten,nine, eight, seven, six or five conservative amino acid substitutions inthe framework region. A variant of a light chain variable regionsequence is identical to the reference sequence except having up to fiveconservative amino acid substitutions in the framework region (i.e.,outside of the CDRs), and preferably has less than four, three or twoconservative amino acid substitution in the framework region.

In another preferred embodiment of the treatment method, medicaments anduses of the present invention, the PD-1 antagonist is a monoclonalantibody which specifically binds to human PD-1 and comprises (a) aheavy chain comprising SEQ ID NO: 14 and (b) a light chain comprisingSEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

In yet another preferred embodiment of the treatment method, medicamentsand uses of the present invention, the PD-1 antagonist is a monoclonalantibody which specifically binds to human PD-1 and comprises (a) aheavy chain comprising SEQ ID NO: 14 and (b) a light chain comprisingSEQ ID NO:18.

In all of the above treatment method, medicaments and uses, the PD-1antagonist inhibits the binding of PD-L1 to PD-1, and preferably alsoinhibits the binding of PD-L2 to PD-1. In some embodiments of the abovetreatment method, medicaments and uses, the PD-1 antagonist is amonoclonal antibody, or an antigen binding fragment thereof, whichspecifically binds to PD-1 or to PD-L1 and blocks the binding of PD-L1to PD-1. In one embodiment, the PD-1 antagonist is an anti-PD-1 antibodywhich comprises a heavy chain and a light chain, and wherein the heavyand light chains comprise the amino acid sequences shown in FIG. 6 (SEQID NO:21 and SEQ ID NO:22).

Table 4 below provides a list of the amino acid sequences of exemplaryanti-PD-1 mAbs for use in the treatment method, medicaments and uses ofthe present invention, and the sequences are shown in FIGS. 1-5.

TABLE 4 EXEMPLARY ANTI-HUMAN PD-1 MONOCLONAL ANTIBODIES A. Compriseslight and heavy chain CDRs of hPD-1.08A in WO2008/156712 CDRL1 SEQ IDNO: 1 CDRL2 SEQ ID NO: 2 CDRL3 SEQ ID NO: 3 CDRH1 SEQ ID NO: 4 CDRH2 SEQID NO: 5 CDRH3 SEQ ID NO: 6 B. Comprises light and heavy chain CDRs ofhPD-1.09A in WO2008/156712 CDRL1 SEQ ID NO: 7 CDRL2 SEQ ID NO: 8 CDRL3SEQ ID NO: 9 CDRH1 SEQ ID NO: 10 CDRH2 SEQ ID NO: 11 CDRH3 SEQ ID NO: 12C. Comprises the mature h109A heavy chain variable region and one of themature K09A light chain variable regions in WO2008/156712 Heavy chain VRSEQ ID NO: 13 Light chain VR SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ IDNO: 17 D. Comprises the mature 409 heavy chain and one of the matureK09A light chains in WO2008/156712 Heavy chain SEQ ID NO: 14 Light chainSEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20

Table 5 provides a brief description of the PD-1 antagonist sequences inthe sequence listing.

SEQ ID NO: Description 1 hPD-1.08A light chain CDR1 2 hPD-1.08A lightchain CDR2 3 hPD-1-08A light chain CDR3 4 hPD-1.08A heavy chain CDR1 5hPD-1.08A heavy chain CDR2 6 hPD-1.08A heavy chain CDR3 7 hPD-1.09Alight chain CDR1 8 hPD-1.09A light chain CDR2 9 hPD-1.09A light chainCDR3 10 hPD-1.09A heavy chain CDR1 11 hPD-1.09A heavy chain CDR2 12hPD-1.09A heavy chain CDR3 13 109A-H heavy chain variable region 14409A-H heavy chain full length 15 K09A-L-11 light chain variable region16 K09A-L-16 light chain variable region 17 K09A-L-17 light chainvariable region 18 K09A-L-11 light chain full length 19 K09A-L-16 lightchain full length 20 K09A-L-17 light chain full length 21 PembrolizumabHeavy chain 22 Pembrolizamub Light chain 23 Nivolumab Heavy chain 24Nivolumab light chain

TABLE 6 Characteristics of Monoclonal Antibody MEB037.22C3 SEQ IDAntibody Feature Amino Acid Sequence NO Light Chain CDRL1KSSQSLLHTSTRKNYLA 55 CDRL2 WASTRES 56 CDRL3 KQSYDVVT 57 Mature VariableDIVMSQSPSSLAVSAGEKVTMTCKSSQSLLHTSTRKNYLAWYQ 58 RegionQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCKQSYDVVTFGAGTKLELKHeavy Chain CDRH1 Kabat Defn SYWIH 59 CDRH1 Chothia Defn GTTFTSYWIH 60CDRH2 YINPSSGYHEYNQKFID 61 CDRH3 SGWLIHGDYYFDF 62 Mature VariableXVHLQQSGAELAKPGASVKMSCKASGYTFTSYWIHWIKQRPG 63 RegionQGLEWIGYINPSSGYHEYNQKFIDKATLTADRSSSTAYMHLTSLTSEDSAVYYCARSGWLIHGDYYFDFWGQGTTLTVSS, wherein X = Q or pE

“PD-L1” or “PD-L2” expression as used herein means any detectable levelof expression of the designated PD-L protein on the cell surface or ofthe designated PD-L mRNA within a cell or tissue. PD-L proteinexpression may be detected with a diagnostic PD-L antibody in an IHCassay of a tumor tissue section or by flow cytometry. Alternatively,PD-L protein expression by tumor cells may be detected by PET imaging,using a binding agent (e.g., antibody fragment, affibody and the like)that specifically binds to the desired PD-L target, e.g., PD-L1 orPD-L2. Techniques for detecting and measuring PD-L mRNA expressioninclude RT-PCR and realtime quantitative RT-PCR.

Several approaches have been described for quantifying PD-L1 proteinexpression in IHC assays of tumor tissue sections. See, e.g., Thompson,R. H., et al., PNAS 101 (49); 17174-17179 (2004); Thompson, R. H. etal., Cancer Res. 66:3381-3385 (2006); Gadiot, J., et al., Cancer117:2192-2201 (2011); Taube, J. M. et al., Sci Transl Med 4, 127ra37(2012); and Toplian, S. L. et al., New Eng. J Med. 366 (26): 2443-2454(2012).

One approach employs a simple binary end-point of positive or negativefor PD-L1 expression, with a positive result defined in terms of thepercentage of tumor cells that exhibit histologic evidence ofcell-surface membrane staining. A tumor tissue section is counted aspositive for PD-L1 expression is at least 1%, and preferably 5% of totaltumor cells.

In another approach, PD-L1 expression in the tumor tissue section isquantified in the tumor cells as well as in infiltrating immune cells,which predominantly comprise lymphocytes. The percentage of tumor cellsand infiltrating immune cells that exhibit membrane staining areseparately quantified as <5%, 5 to 9%, and then in 10% increments up to100%. For tumor cells, PD-L1 expression is counted as negative if thescore is <5% score and positive if the score is ≥5%. PD-L1 expression inthe immune infiltrate is reported as a semi-quantitative measurementcalled the adjusted inflammation score (AIS), which is determined bymultiplying the percent of membrane staining cells by the intensity ofthe infiltrate, which is graded as none (0), mild (score of 1, rarelymphocytes), moderate (score of 2, focal infiltration of tumor bylymphohistiocytic aggregates), or severe (score of 3, diffuseinfiltration). A tumor tissue section is counted as positive for PD-L1expression by immune infiltrates if the AIS is ≥5.

The level of PD-L mRNA expression may be compared to the mRNA expressionlevels of one or more reference genes that are frequently used inquantitative RT-PCR, such as ubiquitin C.

In some embodiments, a level of PD-L1 expression (protein and/or mRNA)by malignant cells and/or by infiltrating immune cells within a tumor isdetermined to be “overexpressed” or “elevated” based on comparison withthe level of PD-L1 expression (protein and/or mRNA) by an appropriatecontrol. For example, a control PD-L1 protein or mRNA expression levelmay be the level quantified in nonmalignant cells of the same type or ina section from a matched normal tissue. In some preferred embodiments,PD-L1 expression in a tumor sample is determined to be elevated if PD-L1protein (and/or PD-L1 mRNA) in the sample is at least 10%, 20%, or 30%greater than in the control.

As used herein, a “pembrolizumab variant” means a monoclonal antibodywhich comprises heavy chain and light chain sequences that are identicalto those in pembrolizumab, except for having three, two or oneconservative amino acid substitutions at positions that are locatedoutside of the light chain CDRs and six, five, four, three, two or oneconservative amino acid substitutions that are located outside of theheavy chain CDRs, e.g., the variant positions are located in the FRregions or the constant region. In other words, pembrolizumab and apembrolizumab variant comprise identical CDR sequences, but differ fromeach other due to having a conservative amino acid substitution at nomore than three or six other positions in their full length light andheavy chain sequences, respectively. A pembrolizumab variant issubstantially the same as pembrolizumab with respect to the followingproperties: binding affinity to PD-1 and ability to block the binding ofeach of PD-L1 and PD-L2 to PD-1.

“RECIST 1.1 Response Criteria” as used herein means the definitions setforth in Eisenhauer et al., E. A. et al., Eur. J Cancer 45:228-247(2009) for target lesions or nontarget lesions, as appropriate based onthe context in which response is being measured.

“Responder patient” when referring to a specific anti-tumor response totreatment with a combination therapy described herein, means the patientexhibited the anti-tumor response.

“Sustained response” means a sustained therapeutic effect aftercessation of treatment with a therapeutic agent, or a combinationtherapy described herein. In some embodiments, the sustained responsehas a duration that is at least the same as the treatment duration, orat least 1.5, 2.0, 2.5 or 3 times longer than the treatment duration.

“Tissue Section” refers to a single part or piece of a tissue sample,e.g., a thin slice of tissue cut from a sample of a normal tissue or ofa tumor.

“Treat” or “treating” cancer as used herein means to administer acombination therapy of a PD-1 antagonist and CpG-C type oligonucleotideto a subject having cancer, or diagnosed with cancer, to achieve atleast one positive therapeutic effect, such as for example, reducednumber of cancer cells, reduced tumor size, reduced rate of cancer cellinfiltration into peripheral organs, or reduced rate of tumor metastasisor tumor growth. Positive therapeutic effects in cancer can be measuredin a number of ways (See, W. A. Weber, J. Nucl. Med. 50:1S-10S (2009)).For example, with respect to tumor growth inhibition, according to NCIstandards, a T/C≤42% is the minimum level of anti-tumor activity. AT/C<10% is considered a high anti-tumor activity level, with T/C(%)=Median tumor volume of the treated/Median tumor volume of thecontrol×100. In some embodiments, response to a combination therapydescribed herein is assessed using RECIST 1.1 criteria or irRC(bidimensional or unidimensional) and the treatment achieved by acombination of the invention is any of PR, CR, OR, PFS, DFS and OS. PFS,also referred to as “Time to Tumor Progression” indicates the length oftime during and after treatment that the cancer does not grow, andincludes the amount of time patients have experienced a CR or PR, aswell as the amount of time patients have experienced SD. DFS refers tothe length of time during and after treatment that the patient remainsfree of disease. OS refers to a prolongation in life expectancy ascompared to naive or untreated individuals or patients. In someembodiments, response to a combination of the invention is any of PR,CR, PFS, DFS, OR and OS that is assessed using RECIST 1.1 responsecriteria. The treatment regimen for a combination of the invention thatis effective to treat a cancer patient may vary according to factorssuch as the disease state, age, and weight of the patient, and theability of the therapy to elicit an anti-cancer response in the subject.While an embodiment of any of the aspects of the invention may not beeffective in achieving a positive therapeutic effect in every subject,it should do so in a statistically significant number of subjects asdetermined by any statistical test known in the art such as theStudent's t-test, the chi²-test, the U-test according to Mann andWhitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test andthe Wilcoxon-test.

The terms “treatment regimen”, “dosing protocol” and “dosing regimen”are used interchangeably to refer to the dose and timing ofadministration of each therapeutic agent in a combination of theinvention.

“Tumor” as it applies to a subject diagnosed with, or suspected ofhaving, cancer refers to a malignant or potentially malignant neoplasmor tissue mass of any size, and includes primary tumors and secondaryneoplasms. A solid tumor is an abnormal growth or mass of tissue thatusually does not contain cysts or liquid areas. Different types of solidtumors are named for the type of cells that form them. Examples of solidtumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers ofthe blood) generally do not form solid tumors (National CancerInstitute, Dictionary of Cancer Terms).

“Tumor burden” also referred to as “tumor load”, refers to the totalamount of tumor material distributed throughout the body. Tumor burdenrefers to the total number of cancer cells or the total size oftumor(s), throughout the body, including lymph nodes and bone marrow.Tumor burden can be determined by a variety of methods known in the art,such as, e.g. by measuring the dimensions of tumor(s) upon removal fromthe subject, e.g., using calipers, or while in the body using imagingtechniques, e.g., ultrasound, bone scan, computed tomography (CT) ormagnetic resonance imaging (MM) scans.

The term “tumor size” refers to the total size of the tumor which can bemeasured as the length and width of a tumor. Tumor size may bedetermined by a variety of methods known in the art, such as, e.g. bymeasuring the dimensions of tumor(s) upon removal from the subject,e.g., using calipers, or while in the body using imaging techniques,e.g., bone scan, ultrasound, CT or MRI scans.

“Unidimensional irRC refers to the set of criteria described in NishinoM, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya N H, Hodi F S.Developing a Common Language for Tumor Response to Immunotherapy:Immune-related Response Criteria using Unidimensional measurements. ClinCancer Res. 2013; 19(14):3936-3943). These criteria utilize the longestdiameter (cm) of each lesion.

“Variable regions” or “V region” as used herein means the segment of IgGchains which is variable in sequence between different antibodies. Itextends to Kabat residue 109 in the light chain and 113 in the heavychain.

In some embodiments of the above treatment method, medicaments and usesof the invention, the individual is a human and the cancer is a solidtumor and in some embodiments, the solid tumor is bladder cancer, breastcancer, clear cell kidney cancer, squamous cell carcinoma of head andneck, lung squamous cell carcinoma, malignant melanoma, non-small-celllung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer,renal cell cancer (RCC), small-cell lung cancer (SCLC) or triplenegative breast cancer. In some embodiments, the cancer is NSCLC,endometrial cancer, urothelial cancer, squamous cell carcinoma of headand neck or melanoma.

In other embodiments of the above treatment method, medicaments and usesof the invention, the individual is a human and the cancer is a hememalignancy and in some embodiments, the heme malignancy is acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuselarge B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinallarge B-cell lymphoma, T-cell/histiocyte-rich large B-cell lymphoma,follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma(MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1),myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, non-Hodgkin'slymphoma (NHL), or small lymphocytic lymphoma (SLL).

Also, in some embodiments of any of the above treatment method,medicaments and uses, the cancer tests positive for the expression ofone or both of PD-L1 and PD-L2. In still other embodiments, the cancerhas elevated PD-L1 expression.

In one embodiment of the above treatment method, medicaments and uses,the individual is a human, the cancer tests positive for human PD-L1 andis selected from the group consisting of NSCLC, endometrial cancer,urothelial cancer, squamous cell carcinoma of head and neck or melanoma.In one embodiment of the above treatment method, medicaments and uses,the individual is a human, the cancer tests positive for human PD-L1 andis advanced or metastatic melanoma.

II. Methods, Uses and Medicaments

In one aspect of the invention, the invention provides a method fortreating cancer in an individual comprising administering to theindividual a combination therapy which comprises a PD-1 antagonist andCpG-C type oligonucleotide.

The combination therapy may also comprise one or more additionaltherapeutic agents. The additional therapeutic agent may be, e.g., achemotherapeutic other than CpG-C type oligonucleotide, a biotherapeuticagent, an immunogenic agent (for example, attenuated cancerous cells,tumor antigens, antigen presenting cells such as dendritic cells pulsedwith tumor derived antigen or nucleic acids, immune stimulatingcytokines (for example, IL-2, IFNα2, GM-CSF), and cells transfected withgenes encoding immune stimulating cytokines such as but not limited toGM-CSF). The specific dosage and dosage schedule of the additionaltherapeutic agent can further vary, and the optimal dose, dosingschedule and route of administration will be determined based upon thespecific therapeutic agent that is being used. In one embodiment, thebiotherapeutic agent is anti-IL-10 antibody or antigen-binding fragmentthereof.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1,see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen,raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole,vorozole, letrozole, and anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Each therapeutic agent in a combination therapy of the invention may beadministered either alone or in a medicament (also referred to herein asa pharmaceutical composition) which comprises the therapeutic agent andone or more pharmaceutically acceptable carriers, excipients anddiluents, according to standard pharmaceutical practice.

Each therapeutic agent in a combination therapy of the invention may beadministered simultaneously (i.e., in the same medicament), concurrently(i.e., in separate medicaments administered one right after the other inany order) or sequentially in any order. Sequential administration isparticularly useful when the therapeutic agents in the combinationtherapy are in different dosage forms (one agent is a tablet or capsuleand another agent is a sterile liquid) and/or are administered ondifferent dosing schedules, e.g., a chemotherapeutic that isadministered at least daily and a biotherapeutic that is administeredless frequently, such as once weekly, once every two weeks, or onceevery three weeks.

In some embodiments, the CpG-C type oligonucleotide is administeredbefore administration of the PD-1 antagonist, while in otherembodiments, the CpG-C type oligonucleotide is administered afteradministration of the PD-1 antagonist. In another embodiment, the CpG-Ctype oligonucleotide is administered concurrently with the PD-1antagonist.

In some embodiments, at least one of the therapeutic agents in thecombination therapy is administered using the same dosage regimen (dose,frequency and duration of treatment) that is typically employed when theagent is used as monotherapy for treating the same cancer. In otherembodiments, the patient receives a lower total amount of at least oneof the therapeutic agents in the combination therapy than when the agentis used as monotherapy, e.g., smaller doses, less frequent doses, and/orshorter treatment duration.

Each small molecule therapeutic agent in a combination therapy of theinvention can be administered orally or parenterally, including theintravenous, intramuscular, intraperitoneal, subcutaneous, rectal,topical, and transdermal routes of administration.

A combination therapy of the invention may be used prior to or followingsurgery to remove a tumor and may be used prior to, during or afterradiation therapy.

In some embodiments, a combination therapy of the invention isadministered to a patient who has not been previously treated with abiotherapeutic or chemotherapeutic agent, i.e., is treatment-naïve. Inother embodiments, the combination therapy is administered to a patientwho failed to achieve a sustained response after prior therapy with abiotherapeutic or chemotherapeutic agent, i.e., istreatment-experienced.

A combination therapy of the invention is typically used to treat atumor that is large enough to be found by palpation or by imagingtechniques well known in the art, such as Mill, ultrasound, or CAT scan.

A combination therapy of the invention is preferably administered to ahuman patient who has a cancer that tests positive for PD-L1 expression.In some preferred embodiments, PD-L1 expression is detected using adiagnostic anti-human PD-L1 antibody, or antigen binding fragmentthereof, in an IHC assay on an FFPE or frozen tissue section of a tumorsample removed from the patient. Typically, the patient's physicianwould order a diagnostic test to determine PD-L1 expression in a tumortissue sample removed from the patient prior to initiation of treatmentwith the PD-1 antagonist and the CpG-C type oligonucleotide, but it isenvisioned that the physician could order the first or subsequentdiagnostic tests at any time after initiation of treatment, such as forexample after completion of a treatment cycle.

Selecting a dosage regimen (also referred to herein as an administrationregimen) for a combination therapy of the invention depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells, tissue or organ in the individualbeing treated. Preferably, a dosage regimen maximizes the amount of eachtherapeutic agent delivered to the patient consistent with an acceptablelevel of side effects. Accordingly, the dose amount and dosing frequencyof each biotherapeutic and chemotherapeutic agent in the combinationdepends in part on the particular therapeutic agent, the severity of thecancer being treated, and patient characteristics. Guidance in selectingappropriate doses of antibodies, cytokines, and small molecules areavailable. See, e.g., Wawrzynczak (1996) Antibody Therapy, BiosScientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) MonoclonalAntibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach(ed.) (1993) Monoclonal Antibodies and Peptide Therapy in AutoimmuneDiseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003) New Engl.J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J.Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' DeskReference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57thedition (November 2002). Determination of the appropriate dosage regimenmay be made by the clinician, e.g., using parameters or factors known orsuspected in the art to affect treatment or predicted to affecttreatment, and will depend, for example, the patient's clinical history(e.g., previous therapy), the type and stage of the cancer to be treatedand biomarkers of response to one or more of the therapeutic agents inthe combination therapy.

Biotherapeutic agents in a combination therapy of the invention may beadministered by continuous infusion, or by doses at intervals of, e.g.,daily, every other day, three times per week, or one time each week, twoweeks, three weeks, monthly, bimonthly, etc. A total weekly dose isgenerally at least 0.05 μg/kg, 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg,100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kgbody weight or more. See, e.g., Yang et al. (2003) New Engl. J. Med.349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liuet al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al.(20003) Cancer Immunol. Immunother. 52:133-144.

In some embodiments that employ an anti-human PD-1 mAb as the PD-1antagonist in the combination therapy, the dosing regimen will compriseadministering the anti-human PD-1 mAb at a dose of 1, 2, 3, 5 or 10mg/kg at intervals of about 14 days (±2 days) or about 21 days (±2 days)or about 30 days (±2 days) throughout the course of treatment.

In other embodiments that employ an anti-human PD-1 mAb as the PD-1antagonist in the combination therapy, the dosing regimen will compriseadministering the anti-human PD-1 mAb at a dose of from about 0.005mg/kg to about 10 mg/kg, with intra-patient dose escalation. In otherescalating dose embodiments, the interval between doses will beprogressively shortened, e.g., about 30 days (±2 days) between the firstand second dose, about 14 days (±2 days) between the second and thirddoses. In certain embodiments, the dosing interval will be about 14 days(±2 days), for doses subsequent to the second dose.

In certain embodiments, a subject will be administered an intravenous(IV) infusion of a medicament comprising any of the PD-1 antagonistsdescribed herein.

In one preferred embodiment of the invention, the PD-1 antagonist in thecombination therapy is nivolumab, which is administered intravenously ata dose selected from the group consisting of: 1 mg/kg Q2W, 2 mg/kg Q2W,3 mg/kg Q2W, 5 mg/kg Q2W, 10 mg Q2W, 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kgQ3W, 5 mg/kg Q3W, and 10 mg Q3W.

In another preferred embodiment of the invention, the PD-1 antagonist inthe combination therapy is pembrolizumab, or a pembrolizumab variant,which is administered in a liquid medicament at a dose selected from thegroup consisting of 1 mg/kg Q2W, 2 mg/kg Q2W, 3 mg/kg Q2W, 5 mg/kg Q2W,10 mg Q2W, 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 5 mg/kg Q3W, 10 mg Q3Wand flat-dose equivalents of any of these doses, i.e., such as 200 mgQ3W. In some embodiments, pembrolizumab is provided as a liquidmedicament which comprises 25 mg/ml pembrolizumab, 7% (w/v) sucrose,0.02% (w/v) polysorbate 80 in 10 mM histidine buffer pH 5.5.

In some embodiments, the selected dose of pembrolizumab is administeredby IV infusion. In one embodiment, the selected dose of pembrolizumab isadministered by IV infusion over a time period of between 25 and 40minutes, or about 30 minutes.

In one embodiment of the invention, the CpG-C type oligonucleotide inthe combination therapy has the sequence of SEQ ID NO: 45. However,other CpG-C type oligonucleotides having the motifs and sequencesdescribed herein are also suitable for use in the combination therapiesof the present invention. Therefore it should be understood, that anydescription pertaining to the methods or medicaments comprising theCpG-C type oligonucleotide of SEQ ID NO:45 is also applicable to otherCpG-C type oligonucleotides, particularly C59-01-C59-14 (SEQ ID NOs:38-51). For the sake of brevity, this understanding will not be repeatedthroughout. In one embodiment of the invention, the CpG-C typeoligonucleotide in the combination therapy has the sequence of SEQ IDNO: 45, and is administered intratumorally at a dose of from 0.1 to 16.0mg once a week, preferably 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0or 8.0 mg once a week. In another embodiment of the invention, theoligonucleotide of SEQ ID NO: 45 is administered intratumorally at adose of from 0.1 to 16.0 mg once a week for four weeks, preferably 0.1,0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 7.0, or 8.0 mg once a week for four weeks.In a further embodiment of the invention, the oligonucleotide of SEQ IDNO: 45 is administered intratumorally at a dose from 0.1 to 16.0 mg onceevery three weeks, preferably 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 7.0, or8.0 mg once every three weeks. In another embodiment of the invention,the oligonucleotide of SEQ ID NO: 45 is administered intratumorally at adose of 2.0, 4.0 or 8.0 mg once a week for four weeks. In yet anotherembodiment of the invention, the oligonucleotide of SEQ ID NO: 45 isadministered intratumorally at a dose of 2.0, 4.0 or 8.0 mg once a weekfor four weeks, followed by once every three weeks. In one embodiment,the oligonucleotide of SEQ ID NO: 45 is administered until progressionor for up to 12-24 weeks after the first dose. In another embodiment,the oligonucleotide of SEQ ID NO: 45 is administered for a total of 4,5, 6, 7 or 8 doses. In some embodiments, the CpG-C type oligonucleotideof SEQ ID NO:45 is administered twice weekly, once weekly, biweekly,once every three weeks, once a month, or bimonthly.

The optimal dose for pembrolizumab in combination with CpG-C typeoligonucleotide may be identified by dose escalation or dosede-escalation of one or both of these agents. In an embodiment,pembrolizumab is administered at 200 mg Q3W and the oligonucleotide ofSEQ ID NO: 45 is intratumorally administered at a dose of from 1 to 16mg once a week, preferably 1.0, 2.0, 4.0, 8.0 or 16.0 mg once a week. Inone embodiment, a patient is treated with 200 mg of pembrolizumab Q3W onDay 1 and treated with the oligonucleotide of SEQ ID NO: 45 administeredintratumorally at a dose from 1 to 16 mg on Day 1, preferably 1.0, 2.0,4.0, 8.0 or 16.0 mg on Day 1, once a week for four weeks, followed by adose of from 1 to 16 mg, preferably 1.0, 2.0, 4.0, 8.0 or 16.0 mg onceevery three weeks. In one embodiment, the oligonucleotide of SEQ ID NO:45 is administered until progression or for up to 24 weeks after thefirst dose. In a further embodiment, the oligonucleotide of SEQ ID NO:45 is administered intratumorally at a dose of from 1 to 16 mg,preferably 1.0, 2.0, 4.0, 8.0 or 16.0 mg on Day 1, once a week for fourweeks, followed by a dose of from 1 to 16 mg, preferably 1.0, 2.0, 4.0,8.0 or 16.0 mg once every three weeks for nine weeks. In anotherembodiment, the oligonucleotide of SEQ ID NO: 45 is administered untilprogression or for up to 24 weeks after the first dose. In anembodiment, the patient is confirmed to have progressive disease whilereceiving prior anti-PD-1 therapy. In another embodiment, pembrolizumabis administered intravenously and administered until progression or upto 45 weeks.

In another embodiment, a patient is treated with 200 mg of pembrolizumabQ3W on Day 1 and treated with the oligonucleotide of SEQ ID NO: 45intratumorally at a dose of from 1 to 16 mg, preferably 1.0, 2.0, 4.0,8.0 or 16.0 mg on Day 22 once a week for four weeks, followed by a doseof from 1 to 16 mg, preferably 1.0, 2.0, 4.0, 8.0 or 16.0 mg once everythree weeks. In a further embodiment, the oligonucleotide of SEQ ID NO:45 is administered intratumorally at a dose of from 1 to 16 mg,preferably 1.0, 2.0, 4.0, 8.0 or 16.0 mg on Day 1 once a week for fourweeks, followed by a dose of from 1 to 16 mg, preferably 1.0, 2.0, 4.0,8.0 or 16.0 mg once every three weeks for nine weeks. In an embodiment,the patient is anti-PD-1/L1 treatment naive. In another embodiment,pembrolizumab is administered intravenously. In another embodiment,pembrolizumab is administered intravenously and administered untilprogression or for up to 45 weeks.

In some embodiments, the patient is treated with the combination therapyfor at least 24 weeks, e.g., eight 3-week cycles. In some embodiments,treatment with the combination therapy continues until the patientexhibits evidence of PD or a CR.

In a further aspect of the invention, the combination therapy whichcomprises a PD-1 antagonist and a CpG-C type oligonucleotide furthercomprises an anti-IL-10 antibody. In one embodiment of the invention,the anti-IL-10 antibody in the combination therapy is anti-IL 10 hum12G8, which is administered intravenously at a dose selected from thegroup consisting of: 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 4 mg/kg Q3W,5 mg/kg Q3W, 6 mg/kg Q3W, 7 mg/kg Q3W, 8 mg/kg Q3W, 9 mg/kg Q3W, 10mg/kg Q3W, 11 mg/kg Q3W, 12 mg/kg Q3W, 13 mg/kg Q3W, 14 mg/kg Q3W and 15mg/kg Q3W. In another embodiment of the invention, the anti-IL-10antibody in the combination therapy is anti-IL-10 hum 12G8, which isadministered intravenously at a dose of 1 mg/kg Q3W. In a furtherembodiment of the invention, the anti-IL-10 antibody in the combinationtherapy is anti-IL 10 hum 12G8, which is administered intravenously at adose of 3 mg/kg Q3W. In yet another embodiment of the invention, theanti-IL-10 antibody in the combination therapy is anti-IL 10 hum 12G8,which is administered intravenously at a dose of 10 mg/kg Q3W.

In a preferred embodiment of the invention, the anti-IL-10 antibody inthe combination therapy is anti-IL-10 hum 12G8, or an anti-IL-10 hum12G8 variant, which is administered in a liquid medicament at a doseselected from the group consisting of 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kgQ3W, 4 mg/kg Q3W, 5 mg/kg Q3W, 6 mg/kg Q3W, 7 mg/kg Q3W, 8 mg/kg Q3W, 9mg/kg Q3W, 10 mg/kg Q3W, 11 mg/kg Q3W, 12 mg/kg Q3W, 13 mg/kg Q3W, 14mg/kg Q3W and 15 mg/kg Q3W.

In some embodiments, the patient is selected for treatment with thecombination therapy of the invention if the patient has been diagnosedwith NSCLC, RCC, endometrial cancer, urothelial cancer, squamous cellcarcinoma of head and neck or melanoma.

The present invention also provides a medicament which comprises a PD-1antagonist as described above and a pharmaceutically acceptableexcipient. When the PD-1 antagonist is a biotherapeutic agent, e.g., amAb, the antagonist may be produced in CHO cells using conventional cellculture and recovery/purification technologies.

In some embodiments, a medicament comprising an anti-PD-1 antibody asthe PD-1 antagonist may be provided as a liquid formulation or preparedby reconstituting a lyophilized powder with sterile water for injectionprior to use. WO 2012/135408 describes the preparation of liquid andlyophilized medicaments comprising pembrolizumab that are suitable foruse in the present invention. In some embodiments, a medicamentcomprising pembrolizumab is provided in a glass vial which containsabout 100 mg of pembrolizumab in 4 ml of solution. Each 1 mL of solutioncontains 25 mg of pembrolizumab and is formulated in: L-histidine (1.55mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and Water for Injection,USP. The solution requires dilution for IV infusion.

The present invention also provides a medicament which comprises a TLR9agonist and a pharmaceutically acceptable excipient, wherein the TLR9agonist is a CpG-C type oligonucleotide. The CpG-C type oligonucleotidemay be reconstituted in a physiological buffer for intratumoralinjection.

The medicaments described herein may be provided as a kit whichcomprises a first container and a second container and a package insert.The first container contains at least one dose of a medicamentcomprising a PD-1 antagonist, the second container contains at least onedose of a medicament comprising CpG-C type oligonucleotide, and thepackage insert, or label, which comprises instructions for treating apatient for cancer using the medicaments. The first and secondcontainers may be comprised of the same or different shape (e.g., vials,syringes and bottles) and/or material (e.g., plastic or glass). The kitmay further comprise other materials that may be useful in administeringthe medicaments, such as diluents, filters, IV bags and lines, needlesand syringes. In some preferred embodiments of the kit, the PD-1antagonist is an anti-PD-1 antibody and the instructions state that themedicaments are intended for use in treating a patient having a cancerthat tests positive for PD-L1 expression by an IHC assay.

These and other aspects of the invention, including the exemplaryspecific embodiments listed below, will be apparent from the teachingscontained herein.

Exemplary Specific Embodiments of the Invention

1. A method for treating cancer in an individual comprisingadministering to the individual a combination therapy which comprises aPD-1 antagonist and a TLR9 agonist, wherein the TLR9 agonist is a CpG-Ctype oligonucleotide.

2. A method for treating cancer in an individual comprisingadministering to the individual a combination therapy which comprises aPD-1 antagonist, an anti-IL-10 antibody or antigen binding fragmentthereof and a TLR9 agonist, wherein the TLR9 agonist is a CpG-C typeoligonucleotide.3. The method of embodiment 1 or 2, wherein the PD-1 antagonist is amonoclonal antibody, or an antigen binding fragment thereof.4. A medicament comprising a PD-1 antagonist for use in combination witha TLR9 agonist for treating cancer in an individual, wherein the PD-1antagonist is a monoclonal antibody, or an antigen binding fragmentthereof and the TLR9 agonist is a CpG-C type oligonucleotide, andpreferably, the PD-1 antagonist is administered before the TLR9 agonist.5. A medicament comprising a PD-1 antagonist for use in combination witha TLR9 agonist and an anti-IL-10 antibody or antigen binding fragmentthereof for treating cancer in an individual, wherein the PD-1antagonist is a monoclonal antibody, or an antigen binding fragmentthereof and the TLR9 agonist is a CpG-C type oligonucleotide.6. A medicament comprising a TLR9 agonist for use in combination with aPD-1 antagonist for treating cancer in an individual, wherein the TLR9agonist is a CpG-C type oligonucleotide.7. Use of a PD-1 antagonist in the manufacture of medicament fortreating cancer in an individual when administered in combination with aTLR9 agonist, wherein the TLR9 agonist is a CpG-C type oligonucleotide.8. Use of a PD-1 antagonist in the manufacture of medicament fortreating cancer in an individual when administered in combination with aTLR9 agonist and an anti-IL-10 antibody or antigen binding fragmentthereof, wherein the TLR9 agonist is a CpG-C type oligonucleotide.9. Use of a TLR9 agonist in the manufacture of a medicament for treatingcancer in an individual when administered in combination with a PD-1antagonist, wherein the TLR9 agonist is a CpG-C type oligonucleotide.10. Use of a PD-1 antagonist and a TLR9 agonist in the manufacture ofmedicaments for treating cancer in an individual, wherein the TLR9agonist is a CpG-C type oligonucleotide.11. A kit which comprises a first container, a second container and apackage insert, wherein the first container comprises at least one doseof a medicament comprising an anti-PD-1 antagonist, the second containercomprises at least one dose of a medicament comprising a TLR9 agonist,and the package insert comprises instructions for treating an individualfor cancer using the medicaments, wherein the TLR9 agonist is a CpG-Ctype oligonucleotide.12. The kit of embodiment 11, wherein the instructions state that themedicaments are intended for use in treating an individual having acancer that tests positive for PD-L1 expression by animmunohistochemical (IHC) assay.13. The method, medicament, use or kit of any of embodiments 1 to 12,wherein the individual is a human and the PD-1 antagonist is amonoclonal antibody, or an antigen binding fragment thereof, whichspecifically binds to human PD-L1 and blocks the binding of human PD-L1to human PD-1.14. The method, medicament, use or kit of any one of embodiments 1-12,wherein the PD-1 antagonist is MPDL3280A, BMS-936559, MEDI4736,MSB0010718C or a monoclonal antibody which comprises the heavy chain andlight chain variable regions of SEQ ID NO:24 and SEQ ID NO:21,respectively, of WO2013/019906.15. The method, medicament, use or kit of any of embodiments 1 to 12,wherein the individual is a human, and the PD-1 antagonist is amonoclonal antibody, or an antigen binding fragment thereof, whichspecifically binds to human PD-1 and blocks the binding of human PD-L1to human PD-1.16. The method, medicament, use or kit of embodiment 15, wherein thePD-1 antagonist also blocks binding of human PD-L2 to human PD-1.17. The method, medicament, use or kit of embodiment 15, wherein themonoclonal antibody, or antigen binding fragment thereof, comprises: (a)light chain CDRs of SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs of SEQID NOs: 4, 5 and 6; or (b) light chain CDRs of SEQ ID NOs: 7, 8 and 9and heavy chain CDRs of SEQ ID NOs: 10, 11 and 12.18. The method, medicament, use or kit of embodiment 15, wherein themonoclonal antibody, or antigen binding fragment thereof, compriseslight chain CDRs of SEQ ID NOs: 7, 8 and 9 and heavy chain CDRs of SEQID NOs: 10, 11 and 12.19. The method, medicament, use or kit of embodiment 15, wherein thePD-1 antagonist is an anti-PD-1 monoclonal antibody which comprises aheavy chain and a light chain, and wherein the heavy chain comprises SEQID NO:21 and the light chain comprises SEQ ID NO:22.20. The method, medicament, use or kit of embodiment 15, wherein thePD-1 antagonist is an anti-PD-1 monoclonal antibody which comprises aheavy chain and a light chain, and wherein the heavy chain comprises SEQID NO:23 and the light chain comprises SEQ ID NO:24.21. The method, medicament, use or kit of any one of embodiments 1-20,wherein the anti-IL-10 antibody or antigen-binding fragment thereofcomprises the heavy chain and light chain variable regions of SEQ IDNO:32 and SEQ ID NO:33.22. The method, medicament, use or kit of any one of embodiments 1-20,wherein the anti-IL-10 antibody, or antigen binding fragment thereof,comprises: (a) light chain CDRs of SEQ ID NOs: 26, 27 and 28 and heavychain CDRs of SEQ ID NOs: 29, 30 and 31.23. The method, medicament, use or kit of any one of embodiments 1-20,wherein the anti-IL-10 antibody is an anti-IL-10 monoclonal antibodywhich comprises a heavy chain and a light chain, and wherein the heavychain comprises SEQ ID NO:34 and the light chain comprises SEQ ID NO:35.24. The method, medicament, use or kit of any one of embodiments 1-20,wherein the anti-IL-10 antibody is anti-IL-10 hum 12G8, or an anti-IL-10hum 12G8 variant.25. The method, medicament, use or kit of any one of embodiments 1-24,wherein the CpG-C type oligonucleotide consists of:(a) 5′-N_(x)(TCG(N))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z),N_(v) (SEQ IDNO:38) wherein N are nucleosides, x=0, 1, 2 or 3, y=1, 2, 3 or 4, w=0, 1or 2, p=0 or 1, q=0, 1 or 2, v=0 to 89 and z=1 to 20, X₁ and X₁′ areself-complementary nucleosides, and X₂ and X₂′ are self-complementarynucleosides; and(b) a palindromic sequence at least 8 bases in length wherein thepalindromic sequence comprises the first (X₁X₂CGX₂′X₁′) of the(X₁X₂CGX₂′X₁′(CG)_(p))_(z) sequences, wherein the oligonucleotide isfrom 12 to 100 bases in length.26. The method, medicament, use or kit of embodiment 25, wherein x=0,y=1, w=0, p=0 or 1, q=0, 1 or 2, v=0 to 20 and z=1, 2, 3 or 4.27. The method, medicament, use or kit of any one of claims 1-24,wherein the CpG-C type oligonucleotide consist ofTCGN_(q)(X₁X₂CGX₂′X₁′CG)_(z)N_(v) (SEQ ID NO:39), wherein N arenucleosides, q=0, 1, 2, 3, 4, or 5, v=0 to 20, z=1 to 4, X₁ and X₁′ areself-complementary nucleosides, X₂ and X₂′ are self-complementarynucleosides, and wherein the oligonucleotide is at least 12 bases inlength.28. The method, medicament, use or kit of any one of embodiments 1-24,wherein the CpG-C type oligonucleotide consist of5′-TCGN_(q)TTCGAACGTTCGAACGTTN_(s)-3′ (SEQ ID NO:40), wherein N arenucleosides, q=0, 1, 2, 3, 4, or 5, s=0 to 20, and wherein theoligonucleotide is at least 12 bases in length.29. The method, medicament, use or kit of any one of embodiments 1-24,wherein the CpG-C type oligonucleotide is selected from the groupconsisting of:

(SEQ ID NO: 41) 5′-TCGTCGAACGTTCGAGATGAT-3′; (SEQ ID NO: 42)5′-TCGTTCGAACGTTCGAACGTTCGAA-3′; (SEQ ID NO: 43)5′-TCGAACGTTCGAACGTTCGAACGTT-3′; (SEQ ID NO: 44)5′-TCGAACGTTCGAACGTTCGAATTTT-3′; (SEQ ID NO: 45)5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′; (SEQ ID NO: 46)5′-TCGTAACGTTCGAACGTTCGAACGTTA-3′; (SEQ ID NO: 47)5′-TCGTAACGTTCGAACGTTCGAACGTT-3′; (SEQ ID NO: 48)5′-TCGTAACGTTCGAACGTTCGAACGT-3′; (SEQ ID NO: 49)5′-TCGTAACGTTCGAACGTTCGAACG-3′; (SEQ ID NO: 50)5′-TCGTAACGTTCGAACGTTCGAAC-3′; and (SEQ ID NO: 51)5′-TCGTAACGTTCGAACGTTCGAA-3′.30. The method, medicament, use or kit of any one of embodiments 1-24,wherein the CpG-C type oligonucleotide has the sequence consisting of5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO:45).31. The method, medicament, use or kit of any of embodiments 1-30,wherein the cancer is a solid tumor.32. The method, medicament, use or kit of any of embodiments 1-30,wherein the cancer is bladder cancer, breast cancer, clear cell kidneycancer, head/neck squamous cell carcinoma, lung squamous cell carcinoma,malignant melanoma, non-small-cell lung cancer (NSCLC), ovarian cancer,pancreatic cancer, prostate cancer, renal cell cancer, small-cell lungcancer (SCLC) or triple negative breast cancer.33. The method, medicament, use or kit of any of embodiments 1-30,wherein the cancer is NSCLC, RCC, endometrial cancer, urothelial cancer,squamous cell carcinoma of head and neck or melanoma.34. The method, medicament, use or kit of any of embodiments 1-30,wherein the individual has not been previously treated for NSCLC, RCC,endometrial cancer, urothelial cancer, squamous cell carcinoma of headand neck or melanoma.35. The method, medicament, use or kit of any of embodiments 1-30,wherein the cancer is advanced or metastatic melanoma.36. The method, medicament, use or kit of any of embodiments 1-30,wherein the cancer is acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloidleukemia (CIVIL), diffuse large B-cell lymphoma (DLBCL), follicularlymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiplemyeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplasticsyndrome (MDS), non-Hodgkin's lymphoma (NHL), cutaneous T-cell lymphoma,or small lymphocytic lymphoma (SLL).37. The method or medicament of any one of claims 1-30, wherein thecancer is selected from the group consisting of renal cell carcinoma,non-small cell lung cancer, bladder cancer and colorectal cancer.38. The method, medicament, use or kit of any of embodiments 1-37,wherein the cancer tests positive for human PD-L1.39. The method, medicament, use or kit of embodiment 38, wherein thehuman PD-L1 expression is elevated.40. The method, medicament, use or kit of embodiment 38, wherein thePD-1 antagonist is pembrolizumab, a pembrolizumab variant or nivolumab.41. The method, medicament, use or kit of embodiment 40, whereinpembrolizumab is formulated as a liquid medicament which comprises 25mg/ml pembrolizumab, 7% (w/v) sucrose, 0.02% (w/v) polysorbate 80 in 10mM histidine buffer pH 5.5.42. A medicament comprising pembrolizumab for use in combination with aCpG-C type oligonucleotide of SEQ ID NO: 45 for treating cancer in ahuman individual by a method comprising first administering to theindividual pembrolizumab, followed by intratumorally administering theoligonucleotide of SEQ ID NO: 45 from one to four weeks later,preferably one, two or three weeks later.43. A method for treating a human individual diagnosed with cancer,comprising administering to the individual a combination therapy whichcomprises pembrolizumab and a CpG-C type oligonucleotide of SEQ ID NO:45, and wherein pembrolizumab is administered at 200 mg Q3W and theoligonucleotide of SEQ ID NO: 45 is intratumorally administered at adose of from 1 to 16 mg once a week, preferably at a dose of 1.0, 2.0,4.0, 8.0 or 16.0 mg once a week.44. A medicament comprising pembrolizumab for use in combination with aCpG-C type oligonucleotide of SEQ ID NO: 45 for treating cancer in ahuman individual by a method comprising administering to the individual200 mg of pembrolizumab Q3W starting on Day 1 and intratumorallyadministering the oligonucleotide of SEQ ID NO: 45 at a dose of from 1to 16 mg starting on Day 22, and then once a week for four weeks,followed by a dose of from 1 to 16 mg once every three weeks, preferablywherein the oligonucleotide of SEQ ID NO: 45 is intratumorallyadministered at a dose of 1.0, 2.0, 4.0, 8.0 or 16.0 mg.45. A medicament comprising pembrolizumab for use in combination with aCpG-C type oligonucleotide of SEQ ID NO: 45 for treating cancer in ahuman individual by a method comprising administering to the individual200 mg of pembrolizumab Q3W starting on Day 1 and intratumorallyadministering the oligonucleotide of SEQ ID NO: 45 at a dose of from 1to 16 mg starting on Day 1 once a week for four weeks, followed by adose of from 1 to 16 mg once every three weeks, preferably wherein theoligonucleotide of SEQ ID NO: 45 is intratumorally administered at adose of 1.0, 2.0, 4.0, 8.0 or 16.0 mg.46. A medicament comprising pembrolizumab for use in combination with aCpG-C type oligonucleotide of SEQ ID NO: 45 for treating cancer in ahuman individual by a method comprising administering to the individual200 mg of pembrolizumab Q3W starting on Day 1 and intratumorallyadministering the oligonucleotide of SEQ ID NO: 45 at a dose of from 1to 16 mg starting on Day 22 once a week for four weeks, followed by adose of from 1 to 16 mg once every three weeks preferably wherein theoligonucleotide of SEQ ID NO: 45 is intratumorally administered at adose of 1.0, 2.0, 4.0, 8.0 or 16.0 mg.47. The method or medicament of embodiment 42-44, or 46, wherein theindividual has not been previously treated with anti-PD-1 or anti-PD-L1therapy.48. The method or medicament of embodiment 43 or 45, wherein theindividual is confirmed progressive while receiving prior anti-PD-1therapy.49. The method or medicament of any of embodiments 42-48, whereinpembrolizumab is administered by IV infusion for 25 to 40 minutes orabout 30 minutes.50. The method or medicament of any of embodiments 42-49, wherein atissue section of the cancer is removed from the individual prior toadministration of the combination therapy tested positive for PD-L1expression.51. The method or medicament of embodiment 50, wherein at least 50% ofthe tumor cells in the tissue section tested positive for PD-L1expression by an immunohistochemical (IHC) assay.52. The method or medicament of embodiment 51, wherein the IHC assayemployed the antibody 22C3 to detect PD-L1 expression.53. The method or medicament of any one of embodiments 1-52, wherein theCpG-C type oligonucleotide is a sodium salt with the sequence of5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO: 45), and theoligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.54 The method, medicament, use or kit of any one of embodiments 1-53,wherein the CpG-C type oligonucleotide has a sequence that consists of5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ (SEQ ID NO:42).55. The method, medicament, use or kit of any one of embodiments 1-53,wherein the CpG-C type oligonucleotide is a sodium salt of5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ (SEQ ID NO:42).56. The method, medicament, use or kit of any of embodiments 42-55,wherein the cancer is advanced or metastatic melanoma.57. The method of embodiment 1, wherein the PD-1 antagonist ispembrolizumab and the CpG-C type oligonucleotide has a sequenceconsisting of 5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′(SEQ ID NO:45).58. The method of embodiment 1, wherein the PD-1 antagonist ispembrolizumab and the CpG-C type oligonucleotide has a sequenceconsisting of 5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′(SEQ ID NO:45), andthe oligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.59. The method of embodiment 57 or 58 wherein the cancer is advanced ormetastatic melanoma.60. The method of embodiment 1, wherein the PD-1 antagonist ispembrolizumab and the CpG-C type oligonucleotide has a sequenceconsisting of 5′-TCGTTCGAACGTTCGAACGTTCGAA-3′ (SEQ ID NO:42).61. The method of embodiment 1, wherein the PD-1 antagonist is amonoclonal antibody, or antigen binding fragment thereof, whichcomprises light chain CDRs of SEQ ID NOs: 7, 8 and 9 and heavy chainCDRs of SEQ ID NOs: 10, 11 and 12, and the CpG-C type oligonucleotideconsists of:(a) 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z),N_(v) (SEQID NO:38) wherein N are nucleosides, x=0, 1, 2 or 3, y=1, 2, 3 or 4,w=0, 1 or 2, p=0 or 1, q=0, 1 or 2, v=0 to 89 and z=1 to 20, X₁ and X₁′are self-complementary nucleosides, and X₂ and X₂′ areself-complementary nucleosides; and(b) a palindromic sequence at least 8 bases in length wherein thepalindromic sequence comprises the first (X₁X₂CGX₂′X₁′) (SEQ ID NO:55)of the (X₁X₂CGX₂′X₁′(CG)_(p))_(z) (SEQ ID NO:56) sequences, wherein theoligonucleotide is from 12 to 100 bases in length.General Methods

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al.(1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fuse with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wrightet al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana etal. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

EXAMPLES Example 1: Immunomodulation of Human Cells by C59-08

C59-08 is a sodium salt of oligodeoxynucleotide5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO: 45) with aphosphorothioate backbone.

Human peripheral blood mononuclear cells (PBMCs) were isolated frombuffy coats from two donors with Ficoll-Paque™ PLUS (GE HealthcareBio-Sciences, Pittsburgh, Pa.) using standard separation methods.Isolated PBMCs were washed twice in phosphate buffered saline (PBS)containing 2% fetal bovine serum (FBS), and 2 mMethylenediaminetetraacetic acid (EDTA). The cells were resuspended andcultured in 96-well U-bottom plates at 1×10⁶ cells per well in RPMI 1640containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mLstreptomycin. The cells were cultured in the presence of C59-08 at dosesranging from 0.016 μM to 5 μM or 7 μM control ODN 1040 in a humidifiedincubator at 37° C., 5% CO₂ in final volume of 0.2 mL for 48 hours.Supernatants were harvested and assayed for IFNα2a and IL-10 using MesoScale Discovery human IFNα2a and human IL-10 tissue culture kits(Rockville, Md.).

The results are shown in FIG. 12. C59-08 induces both IFNα2a and IL-10production in human PBMCs with an optimal concentration of 0.2 μM.

Example 2: Immunomodulation of Human Tumor Specimens by C59-08

Human Tumor Histocultures

Human tumor specimens from patients were obtained from commercialsources (Bio-Options, Folio, Coversant Bio, and Boston BioSource) andUniversity of Rochester. Fresh tumor tissues were collected within 1hour following surgery and placed into AQIX transportation media (AQIX,UK). Tissues were transported overnight at 4° C. to Merck ResearchLaboratories, Palo Alto, Calif.

The tumors were embedded in UltraPure™ low melting point agarose(Invitrogen, Carlsbad, Calif.) and were cut 400 μm with Mcllwain™ TissueChopper (Stoelting Co., Wood Dale, Ill.). The tumor slices were firstset on the Millicell-CM cell culture insert (Millipore, Billerica,Calif.) and cultured at the interface between air and medium of 1 mlDMEM supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate(Mediatech, Inc., Manassas, Va.), 10% FBS (SAFC Biosciences, Lenexa,Kans.), 100 U/ml penicillin, and 100 ug/ml streptomycin in humidifiedincubator at 37° C., 5% CO₂.

The tumor slices were cultured in the presence of 0.1, 0.5, and 1 μMC59-08 or 1 μM control ODN 1040 for 24 hours. The tumor samples weresnap frozen in dry ice and stored at 37° C. prior to processing.

RNA Isolation and Real-Time Quantitative PCR

Total RNA was isolated by homogenization into RNA STAT-60 (Tel-Test,Friendswood, Tex.) using a polytron homogenizer. The total RNA wasextracted according to the manufacturer's protocol. After precipitationwith isopropanol, total RNA was re-extracted withphenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, St. Louis,Mo.) using phase-lock light tubes.

DNase-treated total RNA was reverse-transcribed using QuantiTect ReverseTranscription (Qiagen, Valencia, Calif.) according to manufacturer'sprotocol. Primers were obtained commercially from Life Technologies(Foster City, Calif.). Real-time quantitative PCR on 10 ng of cDNA fromeach sample was performed using unlabeled primers at 900 nM each with250 nM of FAM-labeled probe in a TAQMAN™ RTqPCR reaction on the FluidigmBiomark sequence detection system (Fluidigm, Foster City, Calif.).Levels of ubiquitin were measured in a separate reaction and were usedto normalize the data by the Δ-Δ Ct method. Using the mean cyclethreshold (Ct) value for ubiquitin and the gene of interest for eachsample, the following equation was used to obtain the normalized values:1.8^((Ct ubiquitin−Ct gene of interest))×10 ⁴.

Treatment Results

Ex vivo treatment of human tumors with C59-08 induced IFNα-induciblegenes (IFNα2, MCP1, MCP2, OAS2, IP-10, GBP1, ISG-54, MxB, and TRAIL),cytokines (IFNβ, IL-10, IL-12, IL-6, and TNFα), and immune activationmarkers (CD80, CD86, CD40, CD70 and OX40L) in renal cell carcinoma (RC)(n=5), non-small cell lung cancer (NSCLC) (n=3), and bladder (n=1) andcolorectal (n=1) cancer histocultures. Data with a specimen from a RCCdonor are shown in FIG. 13: (A) IFNα-inducible genes; (B) cytokines; and(C) immune activation markers.

Example 3: Anti-Tumor Activity of Combination of Anti-IL-10 andIntratumoral C59-08 in Animal Model

TC40.11D8 is a mouse IgG1/kappa monoclonal antibody targeted againstmouse IL-10. The mouse IgG1 isotype control is a mouse monoclonalantibody specific for adenoviral hexon 25. Both antibodies were obtainedfrom internal sources as frozen (−80° C.) stocks.

Formulations of Antibodies

The formulation buffer is specific for each antibody to stabilizeproteins and prevent precipitation. The formulations for both TC40.11D8and the mouse IgG1 isotype control were 75 mM sodium chloride, 10 mMsodium phosphate, 3% sucrose, pH7.3.

Oligodeoxynucleotides

Cytidine phospho-guanosine (CpG)-based phosphorothioateoligodeoxynucleotide (ODN) CpG 1826 5′-tccatgacgttcctgacgtt-3′ (SEQ IDNO: 53) (InvivoGen, San Diego, Calif.) is a mouse TLR9 specific agonist.CpG 1826 has a CpG-B type sequence. CpG-based phosphorothioate ODNC59-08 (Dynavax, Berkeley, Calif.) is an agonist that activates bothhuman and mouse TLR9. C59-08 has a CpG-C type sequence:5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO:45), wherein the 5′ and3′ is an OH group.

Control ODN (Dynavax, Berkeley, Calif.) has a non-CpG sequence with aphosphorothioate backbone 5′-TGA CTG TGA ACC TTA GAG ATG A-3′ (SEQ IDNO:54).

Formulations of Oligodeoxynucleotides

CpG 1826 was reconstituted in 0.9% sodium chloride at a concentration of2 mg/mL, aliquoted, and stored at −20° C. C59-08 was reconstituted inphosphate buffered saline (PBS) at a concentration of 4.53 mg/mL,aliquoted, and stored at −20° C. Control ODN was reconstituted in PBS ata concentration of 4.47 mg/mL, aliquoted, and stored at −20° C.

Animals

Approximately seven to eight week old female C57BL/6J mice were obtainedfrom Jackson Laboratory (Sacramento, Calif.). Conventional animal chowand water were provided ad libitum. Animals were housed for one weekprior to the start of the study. The average weight of the animals atthe start of the study (i.e. tumor implantation) was 19 grams.

Procedures involving the care and use of animals in the study werereviewed and approved by the Institutional Animal Care and Use Committeeat Merck Research Laboratories. During the study, the care and use ofanimals were conducted in accordance with the principles outlined in theguidance of the Association for Assessment and Accreditation ofLaboratory Animal Care (AAALAC), the Animal Welfare Act, the AmericanVeterinary Medical Association (AVMA) Euthanasia Panel on Euthanasia,and the Institute for Laboratory Animal Research (ILAR) Guide to theCare and Use of Laboratory Animals.

Tumor Cell Line Preparation and Implantation

The TC-1 cell line, provided by Johns Hopkins University (Baltimore,Md.) is derived from mouse primary lung epithelial cells that werecotransformed with human papilloma virus (HPV-16) E6 and E7 and c-Ha.rasoncogene (Lin et al., Cancer Res., 56:21-6, 1996). TC-1 cells aresyngeneic to the C57BL6/J mouse strain.

The TC-1 cells were cultured in DMEM supplemented with 10% fetal bovineserum and 0.4 mg/mL Geneticin. Sub-confluent TC-1 cells were injectedsubcutaneously (SC) in 0.1 mL of serum-free DMEM in both lower dorsalflanks (1×10⁵ in right flank and 0.5×10⁵ in left flank) of each animal.Animals were first shaved with electric clippers in the areas that wereused for the implantation.

Tumor Measurements and Body Weights

Tumors were measured the day before the first dose and twice a weekthereafter. Tumor length and width were measured using electroniccalipers and tumor volume determined using the formula Volume(mm³)=0.5×Length×Width where length is the longer dimension. Animalswere weighed the day before the first dose and twice a week thereafter.To prevent bias, any outliers by weight or tumor volume were removed andthe remaining mice were grouped into various treatment groups based onthe tumor volume in the right flank (referred to as the injected tumor).

Dosing Solution Preparation

Frozen stocks of the antibodies were thawed and transferred to wet ice.To avoid repeated freeze thaw, each vial of stock was thawed once andaliquots made in volumes sufficient for one time use. Polypropylene, lowadhesion tubes were used for this purpose. The aliquots were stored at−80° C. Before each dosing, one aliquot was thawed and diluted tonominal concentration in the appropriate diluent.

Before each dosing, aliquots of the ODNs (control ODN, CpG 1826, andC59-08) were thawed and diluted to nominal concentration in 0.9% sodiumchloride.

Administration of Antibodies and Oligodeoxynucleotides

Isotype control mIgG1 and anti-IL-10 mIgG1 were administeredintraperitoneally (IP) at 10 mg/kg on Days 0, 4, 8, and 12. Control ODN(2.5 mg/kg), CpG 1826 (1 mg/kg), and C59-08 (2.5 mg/kg) wereadministered intratumorally (IT) only in right tumors on Days 0, 4, 8,and 12.

Statistical Methods

Tumor volumes were compared between treatments at each day of follow-up.Follow-up of individual animals could be terminated early because ofexcessive tumor burden or other reasons. Depending on the reason andtumor size at the last measurement, the last observed tumor volume wastreated as a lower bound on volume at all later days for that animal(right-censored data).

To compare two treatment groups on a given day, a generalization of thenonparametric Mann-Whitney (or Wilcoxon rank sum) test that allows forright-censored data was used: the Peto and Peto version of theGehan-Breslow test. Two-sided p-values were estimated from 20,000 randomreassignments of animals between the two treatments being compared. Tocontrol the familywise error rate across all time points for a givenpair of treatments, p-values were multiplicity adjusted by applying themaxT procedure of Westfall and Young to the permutation distributions. Ap-value of less than 0.05 was used to define statistical significance.

For descriptive purposes, volumes for each day and treatment group weresummarized by their median. To allow for censoring, a distributionfunction for each day and treatment group was estimated by theKaplan-Meier method, with confidence band using Greenwood's formula on alog scale. The median was estimated as the 50th percentile of thedistribution function, with confidence interval obtained by invertingthe confidence band. A 68% confidence level was used, to be comparableto the common “mean±SE” format for summarizing data, since the latter isapproximately a 68% confidence interval for the mean.

When follow-up of an animal was terminated early, the reason wascategorized and the animal's data were handled as follows: (1) tumorburden: right-censor at last measured value; (2) tumor ulceration:right-censor at last measured value, provided this exceeded a threshold(1000 mm³); otherwise omit animal at later times; (3) weight loss/ill(including found dead with evidence of illness): omit animal at latertimes; and (4) unrelated to treatment (e.g., accident found dead with noevidence of illness, administrative termination): right-censor at lastmeasured value, provided this exceeded a threshold (1000 mm³); otherwiseomit animal at later times.

Treatment Results

TC-1 tumor-bearing C57BL/6J mice were grouped into 5 treatment groupsthe day before the first dose when the mean volume of tumors on rightflank reached approximately 60 mm³ (39 mm³-87 mm³): (1) mIgG1 isotypecontrol+control ODN; (2) mIgG1 isotype control+C59-08; (3)anti-IL-10+CpG 1826; (4) anti-IL-10+control ODN; and (5)anti-IL-10+C59-08. The range of volumes of tumors on left was 0 mm³-113mm³. Complete regression (CR) of a tumor was defined as the absence of ameasurable tumor at the time the measurement was conducted, given that atumor was measurable on the day that animals were grouped. The resultsare shown in FIGS. 10 and 11. Anti-IL-10 in combination with eitherintratumoral CpG 1826 (Group 3) or C59-08 (Group 5) resulted in CRs ofinjected tumors in at least 3 animals (FIG. 10A). However, onlyanti-IL-10 in combination with C59-08 (Group 5) resulted in CRs (threeof ten animals) of non-injected tumors (FIG. 11A). Other treatmentsincluding C59-08 monotherapy (Group 2) did not result in CRs of eitherinjected or non-injected tumors. Compared to control treatment,anti-IL-10 monotherapy, and C59-08 monotherapy, administration ofanti-IL-10 in combination with C59-08 (IT) resulted in significantlyreduced volumes of injected tumors for Days 6, 9, and 12 (p<0.05,multiplicity adjusted across time points) (FIG. 10B-D). Compared tocontrol treatment and anti-IL-10 monotherapy, administration ofanti-IL-10 in combination with C59-08 (IT) resulted in significantlyreduced volumes of non-injected tumors for Days 6, 9, and 12 (p<0.05,multiplicity adjusted across time points) (FIG. 11B-D).

Example 4: Anti-Tumor Activity of a Combination of Systemic Anti-PD-1Antibody and Intratumoral CpG-C Oligonucleotide

Antibodies.

Two anti-PD-1 blocking antibodies were used: 29F.1A12 in initial studiesand RMP1-14 in later studies. Each dose contained 250 μgantibody/injection. 29F.1A12 is a purified rat anti-mouse PD-1 antibody(Catalog No. 135202) obtained from BioLegend (San Diego, Calif.). TheBioLegend anti-PD-1 antibody is rat IgG2a, kappa monoclonal antibody.Clone RMP1-14 is a purified rat anti-mouse PD-1 antibody (Catalog No.BE0146) obtained from BioXCell Inc. (West Lebanon, N.H.). The BioXCellanti-PD-1 antibody is a rat IgG2a monoclonal antibody.

Oligodeoxynucleotides.

The non-CpG, control oligodeoxynucleotide (CTRL-ODN), has the sequence5′-TGA CTG TGA ACC TTA GAG ATG A-3′ (SEQ ID NO:54) with aphosphorothioate backbone. Each dose contained 50 μg ODN/injection.

Animals and Cells.

Female BALB/c mice of 6 to 8 weeks of age were obtained from HarlanLaboratories (Indianapolis, Ind.). CT26 is a murine, fibroblast cellline (CT26.WT, Catalog No. CRL-2638™) obtained from American TypeCulture Collection (ATCC, Manassas, Va.). CT26 is anN-nitroso-N-methylurethane-induced, undifferentiated colon carcinomacell line that is frequently used as a model to test immunotherapyregimens (Wang et al., J Immunol, 154: 4685-4692, 1995).

Monotherapy Dosing Regimen.

About 8×10⁴ CT26 cells were injected subcutaneously (SC) into the flankof BALB/c mice (n=5 to 6/group) on Day 0, using a previously publishedmethod (Brattain et al., Cancer Res, 40:2142-2146, 1980). Anti-PD-1blocking antibody was injected intraperitoneally (IP) on Days 5, 8, 11,14 and 18.

Combination Therapy Dosing Regimen.

About 8×10⁴ CT26 cells were injected subcutaneously (SC) into the flankof BALB/c mice (n=5 to 6/group) on Day −7 (Brattain et al., supra,1980). Treatment regimens started on study Day 0 (7 days after tumorcell implantation; average tumor length 5 mm). Mice were left untreatedor injected intraperitoneally (IP) with 200 mcg of a mouse anti-PD-1blocking antibody in a volume of 200 μL (neat formulation as provided bythe manufacturer). Anti-PD-1 injections were administered on Days 0, 3,7, 10, 14, 18, 21 and 25. After several anti-PD-1 injections (Day 12),mice were injected intratumorally (IT) with 50 mcg of C59-08 or CTRL-ODNin a volume of 150 μL PBS. C59-08 and CTRL-ODN injections wereadministered on Days 12, 14, 18, 21, 25, and 28. In both groups,anti-PD-1 treatment was continued as described above. A separate groupof mice with similar-sized tumors (tumor cells were injected on studyDay 0) were injected with C59-08 alone on Days 12, 14, 18, 21, 25, and28, in the absence of anti-PD-1 pre-treatment.

Combination Therapy Dosing and T Cell Depletion Regimen.

About 8×10⁴ CT-26 tumor cells were injected SC in the flank of mice onDay 0 (Brattain et al., supra, 1980). Mice were left untreated, ortreated with anti-PD-1 by IP injection on Days 5, 9, 12, 15, 19, 22, 26and 29. After several anti-PD-1 injections (Day 15), mice were treatedwith C59-08 by IT injection on Days 15, 19, 22, 26 and 29, or were leftuntreated. Anti-CD8 or anti-CD4 depleting antibodies were administeredby IP injection on Days 14, 15, 16, 19, 22, 26 and 29 to mice in theanti-PD1/C59-08 treated group. The anti-PD-1 treated mice received 250μg/injection of the BioXCell RMP1-14 antibody. The mice also received 50μg/injection of C59-08. For depletions, mice received 250 μg/injectionof either an anti-CD8 Ab (YTS 169.4) or an anti-CD4 Ab (GK1.5), bothobtained from BioXCell.

Combination Therapy Dosing Contralateral Tumor Rejection Regimen.

About 8×10⁴ CT-26 cells were injected SC in the left flank on Day 0 andon the right flank on Day 2. Mice were either left untreated (n=18), orinjected with anti-PD-1 Ab, IP (n=19) on Days 5, 7, 11, 14, 21, 23 and26. After several anti-PD-1 Ab injections (Day 14), anti-PD-1-treatedmice were injected with C59-08 in the left tumor on Days 14, 19, 21, 23,and 26. The anti-PD-1 Ab treated mice received 250 μg/injection of theBioXCell RMP1-14 antibody, and 50 μg/injection of C59-08.

Tumor Processing for Extraction of Tumor Infiltrating Leukocytes.

Tumors were placed in a petri dish using forceps, and 5 mL of 5% FCS inRPMI media was added. The tumors were cut into small pieces usingscissors, and minced using the bottom of a 3 mL syringe plunger untiltumor tissue could be pipetted with a 50 mL pipet. The tumor tissuesuspension was transferred into a 50 mL tube, and the petri dish wasrinsed using 5 mL of 5% FCS in RPMI media twice. The tissue suspensionwas digested in a 100× tumor digestion enzyme mix containing 50 mg/mLcollagenase 4 (Sigma-Aldrich C5138-100MG collagenase from Clostridiumhistolyticum) and 2 mg/mL DNase I (Sigma-Aldrich DN25-100MGdeoxyribonuclease I from bovine pancreas). Tubes were incubated at 37°C. for 20 minutes with gentle shaking every 3 min. Samples were filteredthrough a 70 μm filter, and the filter was subsequently washed with 5%FCS in RPMI. Samples were centrifuged at 1400 rpm for 7 minutes at roomtemperature. Cells were re-suspended in 1 to 5 volumes of 5% FCS in RPMIdepending on tumor size. Cells in the resulting suspension were countedusing a hemocytometer.

RNA Extraction from Whole Tumors for Gene Expression Analysis.

Whole tumors were frozen in RNAlater (Catalog No. 76104) obtained fromQiagen (Venlo, NL). After thawing, total RNA was isolated from 30 mg oftotal homogenized whole tumor using the RNeasy Mini Kit (Catalog No.74106) from Qiagen according to the manufacturer's instructions.Briefly, whole tumors stored in RNAlater RNA stabilization Reagent werethawed, weighed, and placed in a 2 mL PCRclean Safe-Lock eppendorf tubecontaining a 5 mm stainless steel bead and RLT buffer with BME (700μL/30 mg tissue, up to 1 mL RLT per tube). Tubes were placed in theTissueLyser Adapter Set 2×24, which was operated twice for 2 min at 25Hz. Lysates were centrifuged for 3 minutes at 13,500 rpm. Thesupernatant was subsequently transferred into a new 15 mL tube. RLTbuffer was added as needed to meet the 700 μL/30 mg requirement. About700 μL of lysate was used and the rest was stored at −80° C. One volumeof 70% ethanol was added to the cleared lysate and 700 μL of sample wastransferred to an RNeasy spin column in a 2 mL collection tube andcentrifuged for 1 minute at 13,500 rpm. If the sample exceeded 700 μL,successive aliquots were processed in the same RNeasy spin column, andflow-through was discarded. 3504, of Buffer RW1 was added to the RNeasyspin column and samples were centrifuged. DNase I incubation mix (80 μL:10 μL DNase I stock solution plus 70 μL Buffer RDD) was added directlyto the RNeasy spin column and incubated at room temperature for 15minutes. 350 μL of Buffer RW1 was added, tubes were centrifuged and theRNeasy spin column was transferred to a new tube. Two volumes of 500 μLBuffer RPE were added to the column and centrifuged for 1 minute to washthe column. The RNeasy spin column was transferred to a new 2 mLcollection tube and centrifuged at full speed for 1 min. The RNeasy spincolumn was transferred into a new 1.5 mL collection tube and 45 μL ofRNase-free water (Life Technologies) was added to the column andcentrifuged for 1 min at 13,500 rpm to elute RNA.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction(TAQMAN)

Five (5) μg of eluted RNA was reverse transcribed by using 5× FirstStrand Buffer (Life Technologies), Bovine Serum Albumin (LifeTechnologies), Recombinant RNasin Ribonuclease Inhibitor (Promega,Madison, Wis.), Oligo(dT)15 (Promega), Random Primers (Promega), dNTP(Invitrogen, Carlsbad, Calif.), DTT (Life Technologies) and SuperScriptIII Reverse Transcriptase (Life Technologies) using a MyiQ Real-Time PCRMachine (Bio-Rad). Data were normalized to ubiquitin expression withcycling conditions of 15 min at 95° C., followed by 40 rounds of 15 secat 95° C. and 1 min at 60° C. Quantification of mRNA was performed usingPower SYBR Green PCR Master Mix (Life Technologies). All quantificationand analysis was performed using an Applied Biosystems (Carlsbad,Calif.) StepOnePlus Real Time PCR system using StepOne v2.1 software.Relative gene expression levels were calculated using the followingformula: 1.8^((Avg Ct Ubi−Ct Gene))*100,000.

Tumor Cell Separation with Lympholyte®-Mammal Cell Separation Media.

This procedure was used to separate tumor infiltrating leukocytes (TIL)from tumor cells. The cell suspension obtained from the tumors wasbrought to 7 mL by addition of 5% FCS in RPMI as needed. 7 mLLympholyte®-Mammal Cell Separation Media (Cedarlane, Catalog No: CL5120)was added to a 15 mL conical, centrifuge tube and then 7 mL of the cellsuspension was carefully layered on top. Cells were spun down at 800×gfor 20 min at room temperature without braking. The top layer thatformed was transferred into a 50 mL tube and filled with 5% FCS in RPMImedia to the 50 mL mark. Cells were pelleted at 1800 rpm for 7 min atroom temperature, under maximum acceleration and maximum braking. Themedia was aspirated and the TIL-containing pellet was re-suspended in 1mL 5% FCS in RPMI media. A fraction of the cells (150 μL) were placed in96-well U-bottom plates, pelleted at 2000 rpm for 3 min, and thenre-suspended with 2504, RLT buffer for gene expression assays. Theremainder of the cells were used for FACS analysis, with about 100-1504,of the TIL sample used for each staining panel.

In Vitro Stimulation of Tumor Infiltrating Leukocytes for CytokinesProduction.

About 1.5×10⁵ TIL isolated with Lymopholyte® Mammal Cell SeparationMedia (Cedarlane, Catalog No: CL5120) were stimulated for 3 hours at 37°C. with a Leukocyte Activation Cocktail containing BD GolgiPlug (500×)obtained from BD Biosciences (Catalog No. 550583) with phorbol myristateacetate (PMA), ionomycin and brefeldin A (BFA), or BFA alone (3 μg/mLfinal concentration), in a final volume of 200 μL. Samples were analyzedfor cytokine production by intracellular staining and flow cytometry.

Cell Staining and Fluorescence Activated Cell Sorting (FACS) Analysis

All reagents were kept at 4° C. All washes involved pipetting the platedcell suspensions up and down three times, followed by centrifuging at1800 rpm for 3 min at 4° C., and discarding the supernatant. Cells werepelleted after stimulation for 3 hours and were resuspended in 80μL/well FACS buffer (PBS, 10% FBS, 0.1% sodium azide) for each sample tocreate a cocktail including 2 μL Fc Blocker and 0.5 μg/mL of eachantibody of interest per sample. Samples were incubated at 4° C. for 20min before washing and resuspension in 200 μL/well FACS buffer. To fixthe cells, 200 μL of 1% paraformaldehyde was added to each well, and theplate was incubated in the dark at 4° C. for 20 min for surfacestaining. The cells were then washed, pelleted and resuspended in 300μl/well FACS buffer, and data were acquired immediately using a flowcytometer (LSRII from BD Bioscience).

For intracellular staining, samples stored overnight at 4° C. werepelleted and resuspended in 0.5% saponin buffer in PBS for 10 min at RTto permeabilize the cells. After an additional spin, cells were stainedwith 80 μL of staining mix plus 2 μL Fc Blocker and antibodies ofinterest in 0.5% saponin buffer in PBS for 30 min at 4° C.: 2.5 μLanti-mouse IFN-γ-PE (Tonbo Biosciences, Cat. 50-7311-U100), and 2.54,anti-mouse TNF-α-APC (Biolegend, Cat. 506308). Cells were pelleted andwashed before resuspension in 300 μL of FACS buffer, and data wereacquired immediately acquired using a flow cytometer (LSRII from BDbioscience).

Mice Bearing CT-26 Tumor Nodules Produce a Heterogeneous Response toSystemic PD-1 Blockade.

Tumor nodule sizes were measured 2 days after the last anti-PD-1injection (day 21). FIG. 14A shows that 17 out of 43 (40%) tumorsexhibited a response to anti-PD-1 treatment, with 8 of the 17 tumors(19%) having completely regressed. The remaining 26 out of 43 tumorsexhibited a size distribution similar to that of control (CTRL)untreated mice. That is, 60% of tumors did respond to the PD-1 blockade.As shown in FIG. 14B, tumors that had a response to anti-PD-1 treatmenthave an increased number of tumor infiltrating leukocytes (TILs) ascompared to untreated (CTRL) tumors or tumors that did not respond toanti-PD-1 treatment. Whole tumors, which were harvested 2-4 days afterthe last anti-PD-1 injection were processed for analysis of geneexpression using a TAQMAN assay. The response to anti-PD-1 correlatedwith the level of expression of T cell infiltration and activationmarkers (FIG. 14C) and type I interferon responsive markers (FIG. 14D).

These data demonstrate that the CT-26 tumor model follows a clearbi-modal response: mice capable of producing an antitumor responsedemonstrate a significant control over tumor growth, whereas mice thatare not able to respond to treatment proceed at the same rate of growthas untreated tumors. In addition, these data show a negative correlationbetween tumor volume and expression of T cell infiltration andactivation, and type I interferon responsive genes.

Intratumoral C59-08 Reverses Tumor Escape from Anti-PD-1 Therapy andLeads to Long-Term, Immune-Mediated Control of Tumor Growth.

FIG. 15A shows the mean tumor volume over time, while FIG. 15B-C showsthe long term survival of mice of various treatment groups. IntratumoralC59-08 with continued anti-PD-1 treatment improves the survival rate ascompared to C59-08 or anti-PD-1 monotherapy. This proof of concept studyindicates that the combination of C59-08 with an anti-PD-1 antibody isable to convert non-responders into responders capable of complete tumorrejection.

CD8+ T Cells but not CD4+ T Cells are Required for the Efficacy ofAnti-PD-1 Plus C59-08 Combination Treatment.

FIG. 16 shows that depletion of CD8+ T cells abolishes the efficacy ofanti-PD-1 plus C59-08 combination treatment.

Anti-PD-1 Plus C59-08 Combination Therapy Allows for Rejection ofContralateral Tumors.

FIG. 17 shows that 13 out of 19 mice (68%) survived and rejected bothC59-08-injected, as well as C59-08-uninjected tumor nodules in theanti-PD-1 plus C59-08 treated group. Untreated mice failed todemonstrate a reduction in tumor size or reject any tumors (0%survival). Thus, the anti-tumor response generated by C59-08 plusanti-PD-1 combination therapy is able to eliminate tumors at a sitedistant from the C59-08 injection.

C59-08 in Combination with a PD-1 Blockade Strongly Induces Infiltrationand Activation of Polyfunctional CD8+ T Cells.

At the time of collection, tumors treated with anti-PD-1, were groupedbased on the rate of response. Tumor Volume (mm³) was calculatedaccording to the formula: (width)²×length/2. Tumors classified asnon-responsive to anti-PD-1 demonstrated an average reduction in volumeof about 20% as compared to control oligonucleotide treated tumors. Incontrast, tumors classified as responsive to anti-PD-1 demonstrated anaverage reduction in volume of about 80% as compared to controloligonucleotide treated tumors.

As shown in FIG. 18A-D, C59-08 was shown to strongly synergize withanti-PD-1 to induce CD8 T cell infiltration and differentiation inpolyfunctional cells able to concomitantly produce IFN-gamma andTNF-alpha. In brief, the increase in number and activation status of theCD8+ T cell infiltrate of combination treated tumors is even better thanthat of anti-PD-1-responsive tumors in the absence of C59-08. Thisindicates that C59-08 has the ability to improve anti-tumor responses inboth anti-PD-1 responders, as well as anti-PD-1 nonresponders.

C59-08 Monotherapy is Effective in Reducing Tumor Volume and InducingExpression of Interferon-Stimulated and Inflammatory Genes.

Mice bearing CT26 colon carcinomas were treated intra-tumorally (IT)with C59-08 or a control oligodeoxynucleotide. On Day 25 (3 days afterthe last treatment), the group injected with the CTRL-ODN was euthanizeddue to excessive enlarged tumors. On Day 35 (13 days after the lasttreatment), the group injected with C59-08 was euthanized. At day 35,two of six mice treated with C59-08 rejected the tumor, leaving notissue available for harvest. In these instances, a value of zero wasused for calculating the mean tumor volume. The remaining four of sixtumors nodules were used to extract TILs.

As shown in FIG. 19A-B, C59-08 was able to inhibit tumor growth and toinduce the expression of IFN-stimulated (ISG15, ISG20, IRF7, MX1, IP-10,and IFIT) and inflammatory (MIG, TNF-alpha, and IFN-gamma) genes inleukocytes purified from the treated tumors. This indicates that C59-08is able to induce a long lasting upregulation of a favorable geneexpression pattern in TILs.

Summary

An antibody to the T cell surface PD-1 receptor acts to block the immuneinhibitory pathway that is switched on by cancer cells (Wolchok andChan, Nature, 515:496-498, 2014). Now as described herein, a CpG-C ODNtermed C59-08 was shown to be efficacious when administered alone, or incombination with an antibody to PD-1, in inhibiting the growth ofestablished tumors in the murine CT26 model of transplantable coloncarcinoma. Specifically, C59-08 was able to inhibit tumor growth,increase the number of TILs, and induce a desirable gene expressionpattern. Although, C59-08 given alone was unable to affect tumorrejection, C59-08 was shown to synergize with anti PD-1 treatmentresulting in the rejection of established tumors, and an increase in theduration of relapse-free survival. Strikingly, addition of intratumoralC59-08 to established anti-PD-1 therapy resulted in the conspicuousinfiltration of activated T cells that correlates to tumor rejection.Thus, the combination of an anti-PD1 antibody and a CpG-C ODN has beenshown to be superior in inducing tumor rejection than either of singleagent alone.

Example 5 Phase 1b/2 Trial of Intratumoral C59-08 in Combination withPembrolizumab in Patients with Metastatic Melanoma

Part 1 (Phase 1b Dose Escalation) evaluates 3 escalating dose levels ofC59-08 in patients with metastatic melanoma, and Part 2 (Phase 2Expansion) will consist of expansion cohorts to further evaluateefficacy and safety in specific melanoma populations. The patientpopulations include:

1) Metastatic melanoma patients who are anti-programmed deathreceptor-1/ligand-1 (anti-PD-1/L1) therapy naive;

2) Metastatic melanoma patients with confirmed progressive disease whilereceiving anti-PD-1 therapy.

In both Parts, patients are treated with 200 mg IV pembrolizumab every 3weeks until progression or for up to 45 weeks after the first dose.

In Part 1, starting on Day 1, patients are treated with 4 weekly dosesof C59-08 at 2, 4 or 8 mg followed by 1 dose every 3 weeks untilprogression or for up to 24 weeks after the first dose. C59-08 isinjected intratumorally into Lesion A, the same site used throughout thetrial. If at any point during treatment, Lesion A has completelyregressed, remaining C59-08 injections are given by peritumoralinjection into the site of Lesion A.

In Part 2 in the expansion cohorts, each patient is treated withpembrolizumab in combination with C59-08 using the dose selected fromPart 1.

In Cohort 1 (anti-PD-1/L1 naïve), starting on Day 22, patients aretreated with C59-08 once a week for 4 weeks followed by once every 3weeks for 9 weeks.

In Cohort 2 (progressive disease on anti-PD-1 therapy), starting on Day1, patients are treated with C59-08 once a week for 4 weeks followed byonce every 3 weeks for nine weeks.

In Part 2, C59-08 is injected intratumorally into up to four lesions(Lesion A, Lesion B, Lesion C, Lesion D), and the same site(s) is usedthroughout the trial. If at any point during treatment the injectedlesion(s) have completely regressed, C59-08 is administered byperitumoral injection into the site(s) of the injected lesions.

REFERENCES

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The invention claimed is:
 1. A method for treating cancer in anindividual comprising administering to the individual a PD-1 antagonistand a TLR9 agonist, wherein the TLR9 agonist is a CpG-C typeoligonucleotide having a sequence consisting of5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′ (SEQ ID NO: 45), and wherein thePD-1 antagonist is: (i) a monoclonal antibody, or antigen bindingfragment thereof, which comprises light chain CDRs of SEQ ID NOs: 7, 8and 9 and heavy chain CDRs of SEQ ID NOs: 10, 11 and 12, or (ii) ananti-PD-1 monoclonal antibody which comprises a heavy chain and a lightchain, and wherein the heavy chain comprises SEQ ID NO: 21 and the lightchain comprises SEQ ID NO:
 22. 2. The method of treating cancer of claim1, wherein the CpG-C type oligonucleotide is an oligodeoxynucleotidewith a phosphorothioate backbone.
 3. The method of treating cancer ofclaim 1, wherein the cancer is a bladder cancer, a breast cancer, aclear cell kidney cancer, a head/neck a squamous cell carcinoma, a lungsquamous cell carcinoma, a malignant melanoma, a non-small-cell lungcancer (NSCLC), an ovarian cancer, a pancreatic cancer, a prostatecancer, a renal cell cancer, a small-cell lung cancer (SCLC), or atriple negative breast cancer.
 4. The method of treating cancer of claim1, wherein the cancer is advanced or metastatic melanoma.
 5. The methodof treating cancer of claim 1, wherein the cancer tests positive forhuman PD-L1.
 6. The method of treating cancer of claim 1, wherein thePD-1 antagonist is pembrolizumab.
 7. The method of treating cancer ofclaim 6, wherein the pembrolizumab is administered at a dose of 200 mgonce every three weeks and the oligonucleotide of SEQ ID NO: 45 isintratumorally administered at a dose of from 1 to 16 mg once a week. 8.The method of treating cancer of claim 6, wherein the pembrolizumab isadministered at a dose of 200 mg once every three weeks and theoligonucleotide of SEQ ID NO: 45 is intratumorally administered at adose of from 1 to 16 mg once every three weeks.
 9. The method oftreating cancer of claim 7, wherein the individual is confirmedprogressive while receiving prior anti-PD-1 therapy.
 10. The method oftreating cancer of claim 8, wherein the individual is confirmedprogressive while receiving prior anti-PD-1 therapy.
 11. The method oftreating cancer of claim 7, wherein the oligonucleotide is injectedintratumorally at a dose of 2.0, 4.0, or 8.0 mg.
 12. The method oftreating cancer of claim 8, wherein the oligonucleotide is injectedintratumorally at a dose of 2.0, 4.0, or 8.0 mg.
 13. The method oftreating cancer of claim 7, wherein the individual has not beenpreviously treated for the cancer with an anti-PD-1 therapy or ananti-PD-L1 therapy.
 14. The method of treating cancer of claim 8,wherein the individual has not been previously treated for the cancerwith an anti-PD-1 therapy or an anti-PD-L1 therapy.
 15. The method oftreating cancer of claim 1, wherein the cancer is an advanced ormetastatic melanoma, a renal cell carcinoma, a non-small cell lungcancer, a bladder cancer, or a colorectal cancer.
 16. The method oftreating cancer of claim 1, wherein the CpG-C type oligonucleotide is asodium salt, and the oligonucleotide is an oligodeoxynucleotide with aphosphorothioate backbone.
 17. The method of treating cancer of claim 2,wherein the CpG-C type oligonucleotide is a sodium salt.
 18. The methodof treating cancer of claim 3, wherein the CpG-C type oligonucleotide isa sodium salt, and the oligonucleotide is an oligodeoxynucleotide with aphosphorothioate backbone.
 19. The method of treating cancer of claim 4,wherein the CpG-C type oligonucleotide is a sodium salt, and theoligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.
 20. The method of treating cancer of claim 5, wherein theCpG-C type oligonucleotide is a sodium salt, and the oligonucleotide isan oligodeoxynucleotide with a phosphorothioate backbone.
 21. The methodof treating cancer of claim 6, wherein the CpG-C type oligonucleotide isa sodium salt, and the oligonucleotide is an oligodeoxynucleotide with aphosphorothioate backbone.
 22. The method of treating cancer of claim 7,wherein the CpG-C type oligonucleotide is a sodium salt, and theoligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.
 23. The method of treating cancer of claim 8, wherein theCpG-C type oligonucleotide is a sodium salt, and the oligonucleotide isan oligodeoxynucleotide with a phosphorothioate backbone.
 24. The methodof treating cancer of claim 9, wherein the CpG-C type oligonucleotide isa sodium salt, and the oligonucleotide is an oligodeoxynucleotide with aphosphorothioate backbone.
 25. The method of treating cancer of claim10, wherein the CpG-C type oligonucleotide is a sodium salt, and theoligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.
 26. The method of treating cancer of claim 11, wherein theCpG-C type oligonucleotide is a sodium salt, and the oligonucleotide isan oligodeoxynucleotide with a phosphorothioate backbone.
 27. The methodof treating cancer of claim 12, wherein the CpG-C type oligonucleotideis a sodium salt, and the oligonucleotide is an oligodeoxynucleotidewith a phosphorothioate backbone.
 28. The method of treating cancer ofclaim 13, wherein the CpG-C type oligonucleotide is a sodium salt, andthe oligonucleotide is an oligodeoxynucleotide with a phosphorothioatebackbone.